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Plasmodium Falciparum - Science topic

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I am using primers from literature to detect the k1 allelic family in samples of Plasmodium falciparum. The expected range of product is 120-280bp, however, I have bright amplification around 500bp for some samples.
I'm not sure why this is in some samples while others are working fine
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Lowering the extension time may help.
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I did in silico study (molecular docking) of anti-malarial compounds' candidates that target the falciparin-2 and plasmodium falciparum dihydrofolate reductase-thymidylate synthase proteins and also in vitro study that target the plasmodium falciparum strain 3D7.
Interestingly, the in silico data and in vitro data are not consistent. Why these phenomenon could be happen? Are there any justification to discuss this phenomenon?
Thanks in advance
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Some of the pesky problems that are not addressed by the in-silico analysis are related to the behaviour of the compounds in aqueous solutions: poor solubility of the compounds, aggregation, unspecific interactions with cell surfaces and sample tubes.
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I am currently taking care of Plasmodium falciparum in vitro culture and in order for me to keep them alive, I was wondering how and when should I feed them if you have any protocol to advice me. I have access to complete media and RPMI.
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You can use the method suggested by Silvia Di Santi. You should change the media every 24hr. If the parasitemia is high, split some and keep it below 5%, we usually keep it around 2-3% unless we want to perform any antimalarial assay. Most importantly, you should check the parasitemia level using microscopy every 2-3 days intervals at least and provide fresh RBCs while splitting. For higher parasitemia, you can use inactivated serum or plasma from AB+ donors.
Below is our article for a brief explanation and synchronization method as well:
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Anyone can help me with this trouble shooting of plasmodium falciparum in vitro culture? I've been tried to culture plasmodium for approximately 2 months but they didn't grow well. Everytime I check thin blood smears of my culture, they appear like this (see the picture I attached) . It seems they couldn't invade or infect the red blood cells and stay outside. Please give me some advice about what I have to do so they can grow well and the ruptured schizont can invade RBCs.
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Please what did you later do to get the cells to grow? Please how do I also get P.falciparum culture? I want to test the effect of my research drug on the growth of plasmodium cells in vitro
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I need a person/team familiar with the isolation of apicoplast from Plasmodium falciparum.
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Thanks to you both, Elemaran and Masoumeh.
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Can someone assist me with analysis of sequence variations among Plasmodium falciparum variant with regards to how these variations might affect HRP2 produced by the available variants.
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Yes please Hao Wang
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I am new to PCR. We are working on PCR based amplification of targeted DNA sequence of Plasmodium Falciparum. For that, We are targeting the Ring stage of the parasite. I want to know on which basis should i decide the gene for primer desinging.
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Thanks!! Divya Das and Katie A S Burnette for your response,
I have chosen the ring stage because symptoms of malaria start to appear in this stage.
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I need to collect blood samples for microscopy of P. falciparum. Which tube is best for this? Heparin or plain vacutainer tubes? Will RBC lysis affect detection of the parasite? Thank you.
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It is possible. Mainly gametocytes and ring forms are found in peripheral blood film and heparine tube does not interfere with the morphology these structures.
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I should isolate single Plasmodium falciparum oocysts from mosquito midguts. In order to do that, I have to stain the midguts first. My present protocol is the following
1. a drop of 1% mercurochrome in PBS on a single midgut and incubation for 5 minutes
2. wash 3 times with PBS for 10 minutes each
3. mount on a slide and put a coverslip to flatten the sample for microscopy observation
For some reason, the oocysts are not stained as they should be (looking at pictures from other publications) and it's not possible to distinguish them by the rest of the sample. Everything has the same pink color.
Does anybody have some good detailed protocol to share? Also, my midgut samples (already dissected) are preserved in 70% ethanol. Could this affect the staining? Should I rehydrate them first or it doesn't matter?
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We normally dissect the midguts in 1% NP-40 in PBS to permeabilise the tissue for 30 min followed by the staining with mercurochrome for at least another 30 minutes. Also we only use 0.1% mercurochrome for staining. Hope that helps although my answer comes quite late.
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Transfection of malaria parasites such as Plasmodium falciparum has been a very difficult experiment. Transfection efficiency is very low even through electroporation method. I wonder whether Lipofectamine 3000 reagent would help me transfect my malaria culture more easily. Please help me figure out.
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How was your experiment? Did It work?
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I recently did a whole genome sequence on Plasmodium falciparum (Illumina , NGS). I have the data but i have been stuck at obtaining the sequences for specific genes of interest. Any tools or pipelines i can use for this? CLC Benchwork didnt work as the sequences are too big!
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Harrison - sorry for the very late reply here, but just noticed this question on RG for CLC so I thought I'd answer it. I'm the product manager for CLC Genomics Workbench @ QIAGEN btw.
You shouldn't have any issue downloading data from NCBI/GenBank within CLC, and there's no limit on genome size you can work with, Plasmodium falciparum included. (Actually - the largest genome ever assembled was done using CLC's de novo assembler 😉) which version of CLC Genomics Workbench are you using?
Plasmodium falciparum 3D7 is the reference genome, correct? based on the info at GenBank - this genome corresponds to assembly ID - GCF_000002765.4
With the latest version of CLC GxWB (v12) - this shoudl be really easy to do. From the menus -
  1. Download > Search for Sequences at NCBI. Once the search window opens, then...
  2. Enter in the field "All Fields" parameter - GCF_000002765.4
  3. Click Start Search
The results should be 14 chromosomes. You can either select all of them at once and click "Download and Save" or - if your university or insittution is on limited bandwidth or being throttled by NCBI, you can try downloading them one at a time.
Here's a screenshot:
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I am trying to perform AFM using plasmodium falciparum infected rbcs Using XE7 Park system, non contact mode. However i have no clue on how to differentiate between infected and non infected rbc as the system is not equiped with a microscope
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How big is max scan area? Usually it is about 100 um, depends on the scanner. You can scan such area (not sure about true non-contact mode, perhaps tapping mode (especially - phase contrast) would be more informative), pick some infected cells (should be possible to distinguish with proper scan size in pixels - 1024 and more) and scan them with proper scan size.
Good luck!
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Can anyone explain the procedure of RNA isolation from plasmodium falciaparum using direct-zol miniprep kit (zymo) ? And I need the information about plasmodium falciparum specification/condition ( culture volume, %parasitemia, ) as a starting material to isolate. Thank you
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Hi Irmayani Irmayani,
I do believe you can easily access such info from the manufacturer's website. Kindly find a link below for a copy of the protocol
Best,
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Hello!
I am researching alternative methods to grow large numbers of Plasmodium falciparum gametocytes (for mosquito infections) which are not the traditional tissue culture flasks, and so can be maintained with minimal human labour if possible. Is anyone aware of any of these methods?
Many thanks!
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Hi, there are some automated systems connecting different tissue culture flasks to change media but it's generally lab made systems with pumps... not sure there are any commercials systems available...
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Would like to know the Plasmodium spp. image database that contains the labeled 5 kinds of species (falciparum, vivax, malariae, ovale, knowlesi) in 4 forms (ring, trophozoite, schizont, gametocyte). The image databases having some of the species and forms are also ok! The image number should be large enough to train a neural network (~1000)
Thank you!
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I am about start conducting a research that involves detecting Plasmodium falciparum in human blood samples in a country located in West Africa. How will I know the best mRDT to use for my study? Please feel free to recommend with references (publications).
Thanks
Ola
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Dear Kokouvi,
Thanks for your candid advice.
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Hi I'm a Bioinformatics PhD student at UoL. I would like to chat to, or network with anyone who is knowledgeable on the cellular/molecular biology of P.falciparum or related strains/organisms such as T.gondii.
I have some very strange data/results which from my Bioinformatics/Comp Bio background does not make much sense in biological terms.
Thanks,
David
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Sorry M. A. A. Al- Fatlawi this link does not work. Is it for a PhD position? I'm already a PhD student at the IIB, Uni of Liverpool
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On plasmodb.org proteins like Stevor and Maurer's cleft which are host-parasite interaction proteins which I presume exist on the parasite cell surface membrane appear with GO annotations for the apicoplast organelle (as contain signal peptide).
I do know that these host-parasite interaction proteins as they are trying to evade the host immune system have different amino acid residue sequence behaviours (due to their evolution), and that homologue searching programs/servers like HMMER can pick up on this.
From a functional biological perspective what would the purpose be of these proteins residing in the apicoplast organelle which is predominantly of biosynthesis and export function be?
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You have me confused at many levels. To the main question, apicoplast (an organelle) does not have cell surface proteins. It is the main parasite cells that do have surface proteins; e.g. merozoite surface proteins on merozoites (the blood stage of Plasmodium) used for RBC invasion, and PfEMP on trophozoites and schizonts (which are inside RBCs) for clustering RBC for ultimate destruction.
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According to a few websites including from wikipedia, The organelle is an adaptation that the apicomplexan applies in penetration of a host cell.
Thus how more specifically in cell/molecular biology terms, how is the apicoplast organelle involved in the parasite invasion? Simplified answers only please as my cell biology/molecular biological knowledge is only intermediate.
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Apicoplast apparently is not involved in cellular invasion - perhaps you misread Wikipedia article. It synthesises fats and isoprenoids for cellular metabolism. Those that are actually involved in invasion are apical secretory organelles including micronemes and rhoptries. If you look for the molecular mechanism of invasion, merozoite surface proteins is the answer.
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Hello,
I am looking for a plasmid for episomal expression in Plasmodium falciparum. Currently I am using pHC3 plasmid but no luck yet, also this plasmid has no map so difficult to handle it. A plasmid that has CAM promoter, supports for a complementation study and also has a full map, would be the ideal one. I appreciate a lot for any suggestion or sharing such plasmid if you are currently using it.
Thank you so much !
Surendra
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Hello, Gibson assembly method, were evaluated for constructing P. falciparum vectors.
See attachment for more info.
Good luck!
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I have to amplify HVR-1 of Plasmodium falciparum ...I diluted primary PCR products 5X and 10X for nested PCR... and I got results in both..which one is more acceptable or effective...
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Hi,
in principle Nested PCR is very effective. From this perspective and based on literature you can even dilute higher and should suceed. Therefore the higher dilution should be better as this will also reduce the amount of all other components form the first amplification like Primers etc
Best Sven
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I am trying to set up an experiment with a tightly synchronized P. falciparum culture to detect changes in expression levels of genes that are present during a particular stage of the parasite life cycle. I have attempted sorbitol synchronization and Percoll synchronization (and a combination of these two methods) and so far I've had no luck. My culture is either not synchronized enough, or I've manipulated it too much and inadvertently killed it.
Does anyone have a good synchronization protocol that produces a highly synchronized culture without killing off the culture? Any help would be immensely appreciated!
Thank you!
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Danielle Snider I feel that a good way to do synchronisation is to closely watch your parasites, make slides every 6 hours to judge the population. If population is made of rings trophozoites and schizonts, I would suggest to stick to only one method i.e. sorbitol (5% Sorbitol for 10-15 minutes at RT or 37 ), gently shake tube and spin at low g values. Wash supernatant that is mainly ruptured cells and rbc's multiple times. add fresh blood maintain a hematocrit of 1% and allow parasites to grow at 37 under gassed environment.
Two sorbitol treatments can be given in tandem cycles to achieve a tight synchrony.
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We now know that apicoplast (Trans) membrane proteins can originate from a vesicle and a signal peptide pathway. Normally signal peptide regions are cleaved out of the protein for apicoplast globular proteins and thus I'm assuming apicoplast membrane residing proteins no longer contain these signal peptide regions either? But I read in one paper that proteins targeted to the apicoplast via vesicles can still have these signal peptide regions within them which is confusing.
As the proteins will join the apicoplast membrane via vesicular fusion, will they not have their signal peptide regions cleaved off? so are there still apicoplast membrane proteins which exist in the these membranes with a signal peptide region within their sequence (which also contains a hydrophobic part)?
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good luck dear David L. Murphy
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It is proposed, in order to reduce the osmotic stress within red blood cells after malaria infection, that plasmodium falciparum recjects nucleic acids out of the red blood cell.
Does anybody know which pathways are used for that?
Best,
Julian
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Dear Jude,
Thanks a lot for your reply. As I am no expert in this field and the question came up in one of our group discussions recently, I think, I would still need to wrap my head around the bigger picture before having a scientific discussion, as it is not directly connected to my work. Anyhow, our general question was based on the following literature:
1.) Virgilio L. Lew, Teresa Tiffert, and Hagai Ginsburg,
Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum–infected red blood cells (BLOOD, 15 MAY 2003 + VOLUME 101, NUMBER 10, 10.1182/blood-2002-08-2654.Supported)
2.)Mailin Waldecker, Anil K. Dasanna, Christine Lansche, Marco Linke, Sirikamol Srismith, Marek Cyrklaff, Cecilia P. Sanchez, Ulrich S. Schwarz, Michael Lanzer,
Differential time‐dependent volumetric and surface area changes and delayed induction of new permeation pathways in P. falciparum‐infected hemoglobinopathic erythrocytes (Cellular Microbiology 2017; 19: e12650 DOI 10.1111/cmi.12650)
Best,
Julian
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I am running restriction digests for dhps gene particularly codons 540, 437 and 436 with the enzymes Fok1,Ava2 and mspA1 respectively. I have realized that with each digestion, the samples get cut but the controls (T9-96, 3D7 and IEC513/86 respectively) remain uncut or disappear altogether. Kindly advice on what could be the problem. Thank you.
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Thank you Paul
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i've been trying to grow my plasmodium falciparum in vitro since december last year but it seems that the parasites didn't want to grow. is there any recommendations for me to troubleshoot this problem? i already increase the amount of albumax and i used fresh blood every time i need to add blood into the culture.
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What does it mean by parasites didn't want to grow? Are they growing up to a certain stage and die? Do you see more gametocyetse ?
Please elaborate on your protocol.
Please try using the following protocol for Albumax (the article is attached for your referance)
Incomplete medium
RPMI 1640 - 10.4 g (1 sachet)
1M Hepes - 25 mL
0.1 M Hypoxanthine - 1 mL (0.1 M hypoxanthine - Dissolve 1.361 g of hypoxanthine in 1M NaOH and make up to 100 mL)
Albumax I - 5 g
Milli-Q water (ultra pure water) - 1 L
Filter sterilize the incomplete medium.
When you want to use the medium, prepare the complete medium by adding sodium bicarbonate to a final concentration of 1.77 mM. (3 mL of 5% (wt/vol) sodium bicarbonate to 100 mL of incomplete medium-Filter sterilize the sodium bicarbonate solution)
Also, please make sure that your red blood cells are free of leukocytes. In my personnel experience, some donor RBCs do not support the growth of malaria parasites. When you get RBCs, make sure your donors are not pre-exposed to malaria parasites. Test different lots of RBCs.
What is your starting hematocrit ? Usually, malaria parasites grow well in 5% hematocrit of 5 mL culture. You can start in small 25 cm2 flasks with a non-vented cap.
I assume you gas the cultures after every media change. That is also a necessary requirement for parasite growth.
Also, what is the source of your parasites? Is it cryopreserved parasites? If so, you can try a different lot of cryopreserved parasites. How many parasites (parasitemia) you add to the starting culture? If they are cryopreserved, they should be thawed carefully, with minimal lysis. Otherwise, if you collect parasitized blood from a human donor, you should wash them thouroughl and gently. Also to establish cultures, ring-stage parasites are preferred as late stage parasites are not preferred for infections.
If you have a perfect protocol and still it doesn't work, you may have to check for micoplasma contamination and any other contaminations.
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I am busy with humanized mice for the study of PK/PD of antimalaria drugs. The work is iinvolving, engraftment of mice with human red blood cells (hRBCS), thereafter induce blood stage infection with plasmodium falciparum parasitized RBCs at a specific concentration. Knowing the concentration is important to monitor (by flow/elecron microscope) if the parasites growth reproductively in their artificial host. My question is to know, how to accurately work out this concentration using trucount tubes?
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well said
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Hi everybody.
My question today is about generating free malaria parasite. I would like to know if someone is able to give me a protocol to extract this parasite from RBC,
using saponine ?
using tween (80 or 20) ?
using sorbitol ?
with concentration of ?
and without ultra-centrifugation if it is possible
ps: I have already read MMR 2013 by D. Mattei (chapter 4) so how do you replace Plasmion ?
Thank you
Best Regards
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Following
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I'm hoping someone with experience working with P. falciparum in vitro can help me with this!
I'm starting in vitro work with P. falciparum, and focusing on culturing gametocytes. I'll be working with strain 3D7.
Through my reading I've found that 3D7 is prone to losing it's gametocyte-formation ability when kept in culture for too long. A lot of papers suggest keeping cultures at a low passage number and continually thawing new feeder stocks to circumvent the problem.
My question is, what is considered a 'low' passage number? None of the papers I've read has given an actual number.
Thank you in advance for any help!
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Hi Danielle,
I used this protocol and it worked very well for me.
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I have fresh dried blood spots that i need to extract Plasmodium falciparum parasite DNA for analysis of Antimalarial drugs Resistance markers.I will sequence the DNA thus i required high quality DNA.How do i go about the very first step ?
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I think the link above has comprehensive information to answer your question
best,
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Hi,
We performed an experiment to measure phagocytosis assay with cFDA to stain the Plasmodium-infected RBCs. I just wonder, is cFDA stain the parasite inside RBCs or only stain the RBCs?
Thank you
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Read the paper on : New Method to Quantify Erythrophagocytosis by Autologous Monocytes ;
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Something about the topic:
sample: whole cell lysate of plasmodium falciparum, 5ug/line;
gel: 12% Tris-Hcl SDS gel;
run: 80v for stacking, 130v for running gel;
stain: silverquest stain for total protein stain.
I saw vertical streak of my protein at the bottom area: I could still see the bands, but they were somehow separated.
The rest of proteins were well positioned as continuous horizontal bands.
It is not likely that I overload the well since it is only 5ug/line.
It is not likely that my samples had somewhat precipitation since I centrifuged them and the streaks only showed up at the bottom.
What's happened?
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What do you mean exactly for "vertical streak"?
Could you maybe attach a picture?
If I properly understood you have kinda a vertical spike in the middle of your band. If it is I had the same problem.
Eventually I found out I just badly prepared my running buffer (either the molarity of Tris-HCl was not 1M or the pH was lower than 8.8) so the gel was "weak".
If you haven't still done it just try to prepare fresh buffer making sure molarity and pH are correct.
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I have tried to detect Plasmodium falciparum lactate dehydrogenase directly from whole malaria infected blood using electrochemical impedance spectroscopy without success. I made dilution of the blood with buffer, PBS, and noninfected blood all to no avail. I wish anyone on this platform could be of help with an idea to solve the problem. Thank you
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Thank you. Your first response solved the problem. The concentration of the PfLDH was too high. As soon as made dilution to about 1000 fold the aptamer was able to bind with a corresponding high charge resistance.
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Please can anyone advise sourcing native plasmodium falciparum lactate dehydrogenase (also Pv-LDH). Does someone already have native purified protein(s) available? We are interested in a collaborative project to prepare more protein and / or direct purchase (2 - 10 mg) and looking to action this quickly.
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May be useful to look at this paper:
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Definitive host allows the parasite to reproduce sexually. Plasmodium sexually reproduce in the mosquito, then why it's an intermediate host?
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Many people talk about the mosquito as being a "vector" and avoid calling in an "intermediate host", considering that sexual reproduction occurs in the mosquito. Other people refer to mosquitoes as definitive hosts of Plasmodium.
The late Norman D. Levine did not like the description "final host" in relation to Toxoplasma and Sarcocystis. He said there is nothing "final" about the carnivore host, transmission being ongoing. He preferred "definitive host".
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Can anyone help me know what is the best way for gene knockdown in plasmodium falciparum.
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Hi All,
I am collecting synchronized plasmodium falciparum samples, every 4 hours for the 48 hours life cycle. I would like to stagger the culture so I collection 2 time points, for a total of 24 hours. Can someone please point in me the right direction. I have read the following paper, but I am still left scratching my head as to how I should proceed.
Thank you in advance!!
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If you have synchronized your malaria parasite using sorbitol why were you sstill having to collect every 4 hours? If you have rings, 24 hours later you will have (majority) late stage trophs. Unless you are collecting various stages of rings, trophs and schizonts you shouldn't have to collect so often for a synchronized culture. Please let us know how your experiment turned out. 
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I am planning to do a SNP analysis to determine the presence of drug resistance signatures in Plasmodium falciparum. Is someone having the scripts which i can use do for my variant calling?
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hi see this link can help  you https://www.ncbi.nlm.nih.gov/pubmed/10697834
also this article
 Out of Africa: origins and evolution of the human malaria parasites
Plasmodium falciparum and Plasmodium vivax
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I am trying to resolve plasmodium falciparum protein on a 10% sds GEL. i am using 0.1% saponin to lyse rbcs, further to which i add loading dye and heat @95 C for 10 min. but the bands are not resolving properly.
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Resolution of a protein in SDS gels depends on size of the protein and the percentage of the gel and how long you let the gel run. Refer the attached picture to get an idea of what percentage to make. Use pre-stained ladder to track the resolution of the bands so that you know how long to let the gel run. 
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I have been trying this for weeks but I do not observe transgenic parasites emerge after drug selection. My protocol in brief is as follows:
(1) Designed gRNAs as 5'-G-N19-NGG-3' to be inserted into a pUF1-Cas9 "suicide vector" which kills the essential gene. (NGG is not included in actual gRNA sequence)
(2) Synthetic Donor plasmid containing left and right Homology arms (with tag included) and PAM sequence shielded by mutation.
(3) Circular plasmids are electroporated into Red Blood Cells (310 V, 1575 Ohm Resistance, and 950 uF capacitance).
(4) DNA-loaded Red Cells are infected with Plasmodium falciparum 3D7 for 2-3 generations.
(5) Drug selection (1.5 uM DSM1) is applied which selects only for Cas9 expression. 
Please suggest any flaw in the procedure or suggest any better protocol to do this.
Thanks in Advance.
Arnish
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I am trying to design an ChIP experiment for var genes of Plasmodium falciparum. Any suggestions on what would be the best way to design the primers for ChIP Assay?
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hello..from NCBI , Primer design 
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Hello all !
Would any of you be able to provide cDNA library of Plasmodium falciparum 3D7 under controlled circumstances? Or, are you aware of anyone who works on this parasite, that I could contact?
Of course, i will afford the shipment of the material.
Looking forward to hearing from you.
Best
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Thank you dear @Uday Mohanta for this valuable information.
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I am carrying out experiments to test the antimalarial activity of a range of iron chelators in vitro.
After quantification of parasitemia by flow cytometry, there was a base level of parasitemia present even at the highest concentrations administered, which I think may be due to the presence of gametocytes (still present in smears post-treatment)
I was wondering if there's a relatively simple way to remove the gametocytes.
Thanks in advance for any help!
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Percoll could be used to remove gametocytes - see for example http://www.ajtmh.org/content/59/4/505.full.pdf (depending a bit on how you set up your Percoll gradient and what you want left over, as you may also find yourself co-purifying away the late-stage asexuals if your culture contains these stages).
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Hey guys,
We want to sequence P. falciparum (23 Mb) on a MiSeq with Nextera XT library prep. I have not worked with falciparum before but literature (see link) says that its extremely AT-biased genome makes library prep difficult because the Illumina polymerase does not effectively amplify AT-rich regions which results in uneven coverage. They suggest adding TMAC to the PCR and using Kapa Hifi as polymerase. However the paper is from 2012 and they did not use the Nextera Kit.
So my question is, has anyone experience with sequencing falciparum and/or has experience with tweaking the Nextera Kit.
Thanks a lot for your help.
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 Dear Martin,
I am very interested in the Kapa Hyper Plus Kit. DId you try it? Did it worked?
Also I normally used Kapa HIFI and I wonder whether adding TMAC makes any substantial difference in terms of reducing AT bias in Plasmodium.
Thanks!!
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We are studying Malaria drugs resistance, and we found that the % of early treatment failure in P. falciparum infected patients treated with ACT higher among children below 5 years.
Is this normal or explanable ?
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An echo to Dr. Kopfli
1. These under 5 yr children had relatively little acquired immunity compared with  the older individuals. 
2. Parasite density would be  higher in these young children.
Therefore, parasite clearance time might take longer in these group of children.
This means parasite clearance time at Day 3 is likely to be lower.
         By definition, ETF means inadequate decrease in parasitemia ( i.e parasitemia on day 3 ≥25% of count on day 0.)
 I feel that you have found ≥25% parasitaemina at Day 3.  
Thank you.
Thank you.
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3D7 strain of Plasmodium falciparum.
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You could easily get it from MR4, it is BEI resources now (https://www.beiresources.org/Home.aspx)
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Hi All,
I plan on culturing our p. falciparum parasites soon, and need a large amount of DNA (>10ug). This is much larger than I normally need, and I am not sure of how much culture I should grow up for this amount. I plan on extracting with a phenol/chloroform method. Does anyone have a suggestion on how much pRBC I should use and at what parasitaemia to grow my parasites to obtain such large amounts (and hopefully in a relativity short time frame)?
Thank you for your thoughts!!!
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Hi Marina,
Good choice of extraction methodology, phenol-chloroform will give you the cleanest and best quantity of DNA. However, your yield will depend on a bunch of factors such as:
  • what life cycle stage you plan to collect samples at - later stage parasites (schizonts) will yield higher quantities of DNA
  • what parasitemia you plan on culturing the parasites at - higher the parasitemia lower can be the total culture volume
  • what hematocrit you plan on maintaining in the cultures - 5% parasitemia at 5% hematocrit will yield greater quantity of DNA than 5% parasitemia at 2% hematocrit
Having said that, on an average I get about 5-7ug of DNA from a 20mL culture of 3-4% parasitemia and 5% hematocrit using phenol-chloroform.
Hope this helps! Good luck!
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We are currently doing Plasmodium falciparum cultures (NF54 strain) in a perspective to infect Anopheles gambiae by membrane feeding. The Culture evolves up to gametocyte (stage V), but we are struggling to get positive infection by membrane feeding.
We get nice gametocytes in (Stage V) but they seem no infectious for the mosquitoes.
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Yes: if the gametocytes are not able to exflagellate (i.e. produce mature male gametes) then there will be no mating with female gametes, no zygotes produced and hence no infection of mosquitoes.  Efficient exflagellation is not a guarantee of efficient mosquito infection, but it's a first step!  Good luck...
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Im using in situ cell death detection kit-fluorescein roche for apoptosis detection in erythrocytic stage of Plasmodium falciparum. I used different protocols, but the problem is with fixation process, where I tried 4% paraformaldehyde as it is recommended, but the cells destroyed immediately after treatment with 0.1% triton X-100!!!!! I also tried 70% ice cold ethanol, but the blood sample clotted !!!! then I used 4% formadehyde with 0.1% glutaraldehyde, it gave some interesting result, but still some green background which should not be observed !!!!
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two more papers attached
joshua
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how can i isolate plasmodium spp. from whole blood?
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For parasite culture Marias answer should be sufficient. If you want to perform down stream analysis such proteomics or transcriptional analysis you may  need to mature the parasites ex vivo in order to capture all the stages (rings, trophs and schizonts). They have unique profiles which you need to be aware of.
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Why we use Albumax during the preparation of complete RPMI media for P. falciparum and what is the function of albumax ?
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Albumax 2 is used as a substitute for human AB+ serum. Albumax is preferred because it is easier than working with human serum as it needs to be filtered and heat inactivated. 
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So far, I have only seen evidence in avian malaria, but melanization, not total clearance; see:
Hillyer, J.F. et al. (2003) Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti. J. Parasitol. 89, 62–69
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I am glad that Dr Beier answered this question because I used his protocol, optimized at the MRTC, Mali, for my csp ELISA.
However, I am not sure I understand what is addressed in Lillian's question.
After the 1st infective blood meal, are we searching for live sporozoites in the salivary gland (ie like dissecting the gland and looking for sporozoite using a stain under the microscope in the case of experimental infestation) or are we searching, using tools and techniques like ELISA, for antigenic determinants of PF, evidence of a prior infection or the potential to have infected earlier through subsequent feeding? 
I may be wrong here but let me just share with you my thoughts and some observations I personally made at the field during mosquito collections.
From parasites biology, for survival purposes, the parasite moves to the point of exit (salivary glands) in a stage infective (sporozoites) to the next host (vertebrate) and induces feeding behaviour in its vectors to enable it continue its development. Once the sporozoite matures, it exits into a vertebrate host and continues its developmental process. Eco-physiologists will call it SURVIVAL STRATEGIES.
I think there is no time of dormancy, reduced activity or waiting as in storage in the salivary glands by sporozoites. This is where the biology of parasite and the biology of vector synchronize into a non consenting agreement to insure parasites survival. 
From the insect biology standpoint, I have never asked myself if there is a limit in the salivary glands for sporozoite load but I believe nature regulates things such that there is no overcrowding of sporozoites in salivary glands. There must be a regulatory mechanism to slow parasite development or enhance exit of sporozoites from the salivary glands. Hence this induced feeding strategy for exit  to the vertebrate host that regulates the gonotrophic cycle in insects.
In case there is no specific host, the insect feeds on any alternative host to free the gland pouch, the space that is needed for the next maturing sporozoites. This is the basis for host substitution as a vector-borne disease control method that has proven effective among rural dwellers in Yoruba land where housing patterns are the" face-me- I- face- you" type with corridors where domestic animals (companion and livestock) pass the night. By this process, anthropophilic vectors gradually develop a zoophilic behaviour. This was also observed among the fulani herdsmen who spend the night covered amid their herds, insects end up exhibiting animal and human blood meal sources.
Finally, I think that if an insect bites once and picks up gametocytes that all develop into sporozoites which exit into the vertebrate host, no live sporozoite will be left if there is no intake of a new batch of gametocytes to develop into fresh sporozoites.
so Clearance of live sporozoites in the case of a single infection is possible but this is impossible in the case of recurrent infections (like in endemic areas) although evidence of infection/ infectivity is once after the first infection via the everlasting presence of antigenic determinants.
I hope I did not confuse you the more Lillian
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can i get commercial plasmid for transfection ? 
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Dear All,
I'm doing the in vitro culture of Plasmodium falciparum (iRBC) with Albumax-I, would like to estimate the phagocytic activity of THP-1 derived macrophages towards iRBC, the literature which I have found were opsonized the iRBC with homologus serum to estimate the phagocytic activity but I am using variable blood groups to grow the culture I couldnt found the homologous serum in this context do the opsonization require or can I directly incubate the pre stained iRBC along with macrophage to estimate the phagocytosis by FACs
Thank you
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Dear all thanks for your suggestions and sparing valuable time for me
Dear Donald thanks for the article 
I have question that from the literature I came to know that plasmodium infected RBC inhibits the phagocytic activity of monocytes my interest of experiment is to enhance the phagaocytic activity by using exogenous chemicals invitro  for this I would like to check the amount of P. falciparum phagocytosis by THP-1 derived macrophages in presence and absence of exogenous chemical kindly suggest the feasible experiment to perform phagocytosis and to estimate the amount of phagocytosis.
*when I checked literature they suggested to use fresh autologous serum for phagocytosis (Is it from malaria patient?) I dont have access to patient serum so kindly suggest the alternate and I am using combination of fresh ABO blood groups for parasite invasion
Thank you
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I am trying to get my plasmodium falciparum parasites synchronized to a 1hr time frame. I do a treatment of 5% sorbitol at 15hpi, then repeat at 5hpi. At 48hpi I run them through a MACs column to purify bursting schizonts, allow to grow for 1 hour and purify with sorbitol to get young rings. It seems as though my sorbitol treatments are too efficient because I still have schizonts and trophs in my culture when I should only have the young rings. Any have suggestions on how to make my window of synchronization tighter without putting stress on the parasites? Thank you in advance!
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Hi Marina,
I was going to recommend the same paper by Boyle et al. as Praveen has already suggested. Check out their supplementary information on heparin synchronisation method. Essentially, by adding heparin into your culture, it'll inhibit invasion. So, your 1 h window synchrony can be achieved by adding heparin into your culture until you want them to start invading. You then wash heparin off and leave them in heparin-free medium for an hour before you put the heparin back in. The tip I can give you is that you should start with quite synchronised parasites using sorbitol treatment at relatively high parasitemia. I'd recommend 4-5%. Since you will only allow a portion of parasites to invade within an hour, it'd be difficult to get a decent amount of parasites in one-hour window if you don't start with high parasitemia culture. Please also be sure to time your parasites right when you need to wash the heparin off (at very late-schizont stage of course when you start seeing some parasites beginning to rupture), otherwise you may lose them all if they have all been ruptured while you still have heparin in the medium. Delaying them at room temp to adjust the timing can also be done. If you cannot find the heparin solution as they suggested in their paper, you can use heparin salt from Sigma (H-3149) and use it at 100 ug/mL final concentration. You may want to try different concentrations if it doesn't work well for you at this concentration (i.e. it doesn't completely inhibit invasion).
Sorry for such a long answer but hope it helps. I've been using this method for years so  please feel free to contact me should you need further explanation. 
Cheers.  
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We have the DNA sequence, and the DNA extract of Plasmodium falciparum from which the DNA was sequenced.
We need the pure protein from the sequences fro immunological work. We lack the technology to express the proteins. Can anyone give us help?
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Hi Nyingchu,
You can also use the bacterial expression vector pET28a(+) with T7lac promoter, which has an N-terminal His tag and kanamycin resistance. You could, for example, use the restriction sites XhoI and NcoI to clone your construct into the expression vector and express it, for example, in KRX cells. But before, make sure that these restriction enzymes do not cut your own construct. For more restriction sites you can use the information about the vector in the internet. Via the His tag you can purify the protein using hexahistidyl affinity chromatography on Ni-NTA-material. Cloning and expression is a daily routine in our lab. Do you have facilities for the expression of proteins and their purification in your own lab? If you have further questions, feel free to aks me...
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Im trying to measure lipid peroxidation using fluorescent probe BODIPY FL C16 (Molecular Probes) using flow cytometry in plasmodium falciparum infected erythrocytes. But Im having troubles analyzing my results. I found that both green and red fluorescence decrease after my induction treatments comparing to control, but my intensity ratios increase. I found in literature ambiguous ways to analyze same data. Some just analyze an increment in green/red ratio, others look for an increase in green fluorescence and others looks for a decrease in green fluorescence. Im a little confused. If anyone has some experience with this dye and any suggestions they will be well received.
Thanks in advance!
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which marker for lipid peroxidation estimation you used 
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I have done whole transcriptome sequencing for Plasmodium falciparum (2 strains) and it is compared. Can anyone tell me how to analyze the data completely, like based on what and how to interpret the result? Thanks in advance.
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Its depends on the objective of your study. Are you looking for annotation or gene expression analysis.
If you are looking for prokaryotic NGS genome annotation follow the below steps.
Get the gDNA raw data
Assembly using VELVET or SOAP de-Denovo
CDS prediction
Fucntional annotation of contigs/CDS using RAST server or Manual BLAST
GO anlaysis
Gene Ontology(GO) mapping for CDS
Biological annotation of individual genes and corresponding CDS
For  gene expression analysis try following steps.
1. Get raw data fastq files.
2. QC using FASTQC or readQC or Groomer tool
3. Trimming filtering using trimmomatic or qtrip or prinseq
4. get reference genome and make Alignment using TOPHAT or Bowtie.
5. Then use gtf file of corresponding genome then transcript assembly and Gene expression analysis using cufflink, cuffcompare, cuffmerge, cuffdiff etc
6. Differential expression analysis using R BIoconductor. Use CummeRbund to analyse cuffdiff results further. CummeRbund is an R package that is designed to aid and simplify the task of analyzing Cufflinks RNA-Seq output.
Read the article by the authors of Tophat and Cufflinks at
Best!!
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I would like to co-culture Plasmodium falciparum (pf) with mice cell line in this case will infection occur or can I separate the pf antigen for infection to mice cell line
Thank you
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It is tough to culture this of parasite in mouse as these parasite are species specific and highly adapted to human cells p. bergi may be a choice for rodent cell line the media which they use for the of culture is RPMI media  but the cells in mouse may not support pf 
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What is the gene of target and appropriate primers for dtection of drugs resistance in plasmodium falciparum?
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As artemether is an artemisinine derivative, we can suggest that they have the same molecular target (the K13 propeller domain). But some studies related to the artemether-lumefantrine combination (studied together) shows some Pfmdr1 copy number increase. 
And finally, some years ago, the PfAtpase6 gene were incriminated too. 
In conclusion, actually, mutations on the K13 propeller gene are the best molecular marker admitted by (I suppose) everybody...
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We recently received some Plasmodium falciparum sporozoites cryovials which are frozen with HES (hydroxy ethyl starch),Trehalose and DOPC (Dioleoylphosphatidyl-Choline) based freezing solution. As we have no experience with these freezing solution, can I please ask if anyone can advice us what principle should we follow when we thaw these cells (also would be fantastic if anyone can provide us a thawing protocol for similar conditions)?
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I do not know your application but I have used the trehalose based freezing solutions with human epithelial cells.  We thawed the cells the same way with any cryo-media, Fast.  Threhalose is not considered toxic.  Similar to raffinose or other branched carbohydrates.
General procedure:
1. Vials were swirled by hand in a 38 C water bath. 
2.  As soon as the lat remnant of ice disappears, we very gently diluted the crryo-media 10 fold
3. cells allowed the cells to rest for 30mins-1 hour in aerobic conditions. 
The thaw is the highest stress point for cells in the cryopreservation procedure.  Respiration restarts and membranes have to be repaired.  Between the damaged membranes and the superoxide generation, not all cells will make it out of cryo.
Hope this helps
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Can Seryl tRNA synthetase of plasmodium falciparum 3d7 be taken as a target drug?
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thanks a lot sir for providing me the information
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Hello, I am planning to carry out an experiment involving Plasmodium falciparum infected Anopheles mosquito. For this, I have to artificially infect the mosquito with human blood containing gametocytes, after collecting the blood in to heparinized tube. So, in what condition should i keep the sample to prevent exflagellation of the microgametes for successful infection of the mosquitoes and for how long can I keep it before carrying out the experimental infection? 
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I like both answer and are right.
Thanks you both
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I am trying to amplify long genes (6Kb-12Kb) using cDNA as template. The RT I used was the SuperScript III with cDNA synthesis capacity is up to 12Kb, the template is an AT-rich genome.
I did check the RNA quality by measuring the 260:30 and 260:28 ratio, and is ok from 1.8 to 2.0. I treated the RNA with DNaseI previous to the RT and I tested by qPCR to confirm the cDNA synthesis using a housekeeping gene.
I did use one HiFi TaKaRa Taq polymerase which theoretically amplifies long fragments. I optimized the PCR protocol on genomic DNA and I confirm the amplification of the large fragments of my interest, now when I try the same protocol used on genomic DNA I do not get amplification.
Does anybody have experiences in long rage PCR using cDNA as template? Can somebody help me with suggestions?
Thanks a lot, those experiments constituted the last ones to finish my thesis and I don’t want to be stucked in this rut.
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 dear petre Ianakiev I need a suggestion as well about my cDNa amplification. I have to amplify a gene of 4000bp with specific primers in order to insert restrction enzyme sequence at the 5' and 3'. I started to RV the RNA and then I tried to amplify the cDNA with the Q5 high fidelity polymarase but I didn't see any band. as above there are larger sequence than this, so I think that is not impossible to amplify a such sequence. Can you or anyone else give me a suggestion? 
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I am carrying out a research project to investigate the dominant circulating variants of the pfAMA1 gene in a population. I have already extracted the plasmodium falciparum gene but need to carry out PCR and sequencing to identify frequency of the different variants. I however need to know the region which exhibits the 3 variants and the length in base pairs so as to perform nested PCR and also determine the cost of sequencing according to the number of base pairs thus preventing wastage
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Dear  Shillah
may I ask why you are only interested in these haplotypes?
For a suitable paper listing a sequencing strategegy for ama1 you could try
Polley and Conway Genetics. 2001 Aug;158(4):1505-12.
although as this paper shows it's been a while since I worked on population genetics of ama1
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What strain of chloroquine resistant  plasmodium falciparum is widely used for research please suggest??
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Ya, totally support using of Dd2 strain for CQ-resistance.
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I am trying to estimate the risk of P falciparum at the community level. I am aware of what has been done globally and regionally. Are there any changes or benefits of doing this at a smaller geographical area level?
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I would think it might be valuable if done as part of a control program. It might allow you to target interventions or identify areas where interventions have not been used adequately (e. g. perhaps hot spots have inadequate bednet usage). 
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I'm doing a proposal for a research to identify the target and mode of action of one drug against P. falciparum using OMICS and one of the strategies is to create a resistant strain from a sensitive one against that drug and check where the mutations occurred on the genes using omics and would like some advice about recommended techniques and protocols.
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Thanks Beatrice
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Hi,
I'm working with RNA extraction from Plasmodium falciparum samples. I want to send the culture sample for my friend. Is it best to store the sample at TRIzol or RNA later and also for transportation?
Please help me with this. Thanks in advance.
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For transportation the RNA is suitable. Since your sample is stored in TRIZol could be dangerous for the transporter and the people around. And in RNA you only have to seal properly the tubes with parafilm.
Regards
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I am isolating Plasmodium falciparum merozoite from 3D7 culture, I am doing sorbitol synchronisation and enrichment through MACS column, followed by maturation of parasite in mature schizont, Than I am passing mature schizont through 1.2 um syringe filter. Every steps are going fine, I am able to get merozoite at the end but it is in ~1 ml volume which is higher enough I want to concentrate in 50-100 ul volume. I tried multiple time to do this by spinning at different speed (up to 10000 rpm) but not successful and I lost larger proportion of merozoite by this process. Can any one share their protocols, even if it is different method. Or if there is optimised protocol that can be used to concentrate merozoite.
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Hi,
Our merozoite purification protocol is published in the open access publication "Methods in Malaria Research'  at MR4
We also published a detail protocol here:
There is a protocol in the supplementary material
We generally avoid high speed centrifugation
I hope these help
Cheers
James
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Up to my knowledge, P. falciparum dhfr mutant alleles are selected in a step-wise fashion, starting with a mutation at codon 108 from serine to asparagine (S108N) whereas that at codon 51 is a successive one. However, I do not know whether 51I could be existent alone without 108N. I would appreciate experts in the field for giving their input about this point.
Best regards,
Rashad
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Artifical mutant alleles have been made with the single mutant forms of dhfr in various systems. It seems the 51 single mutant form of dhfr does not confer resistance to the antifolates - and therefore would confer no advantage to the parasite carrying it, and it would not be selected. In addition the N51I mutation seems to affect the Kcat of the enzyme, which could affect the fitness of the parasite in the absence of antifolate drugs as well.
So we don't see the single mutant because although it could occur by random mutation, the parasite carrying the mutation would have no selective advantage in the presence of antifolates, and would also be worse off in the absence of drug. The parasite would therefore not outcompete wild-type (or other mutant forms), and would either remain at the very low frequency it started at, be lost by genetic drift, or may even not survive at all if the fitness cost in the absence of drug is significant.
These papers may be useful in providing the detail:
CHUSACULTANACHAI, S., THIENSATHIT, P., TARNCHOMPOO,
B., SIRAWARAPORN, W. & YUTHAVONG, Y. (2002). Novel
antifolate resistant mutations of Plasmodium falciparum
dihydrofolate reductase selected in Escherichia coli.
Molecular and Biochemical Parasitology 120, 61–72.
FERLAN, J. T., MOOKHERJEE, S., OKEZIE, I. N., FULGENCE, L.
& SIBLEY, C. H. (2001). Mutagenesis of dihydrofolate
reductase from Plasmodium falciparum: analysis in
Saccharomyces cerevisiae of triple mutant alleles
resistant to pyrimethamine or WR99210. Molecular and
Biochemical Parasitology 113, 139–150.
HANKINS, E. G., WARHURST, D. C. & SIBLEY, C. H. (2001).
Novel alleles of the Plasmodium falciparum dhfr highly
resistant to pyrimethamine and chlorcycloguanil, but
Malarial DHFR-TS as antifolate target 257
not WR99210. Molecular and Biochemical Parasitology
117, 91–102.
SIRAWARAPORN, W., SATHITKUL, T., SIRAWARAPORN, R.,
YUTHAVONG, Y. & SANTI, D. V. (1997b). Antifolateresistant
mutants of Plasmodium falciparum
dihydrofolate reductase. Proceedings of the National
Academy of Sciences, USA 94, 1124–1129.
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I have a stock of RBC which is more than one month old but still my parasites are growing well in them as observed using giemsa staining. I'm planning for stable transfection. So can I use the same RBC (after washing step) or should I use fresh RBC.
Does change in batch of RBC change the growth of parasites.
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We always transfect into fresh RBCs (certainly less than 2 weeks from blood draw), even if the parasite look like they're growing ok in older blood.  For a transfection, you want your parasites growing optimally and they are generally acknowledged to grow best in freshly-drawn blood because, as Praveen notes, blood cells age over time and parasites grow in the youngest cells most efficiently.  We also 'feed' them weekly after transfection for the same reason (cells age even faster at 37C than when stored in the fridge).  Because blood batches do differ, Kunal's suggestion to mix a couple of different batches may also help your transfections to succeed.
See reference on optimal transfection procedures:
'A quantitative analysis of Plasmodium falciparum transfection using DNA-loaded erythrocytes.'
Hasenkamp, S., Merrick, C.J., Horrocks, P. Molecular and Biochemical Parasitology 187(2), 117-120 (2013).
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Several approaches are available for the molecular detection of P. falciparum gametocytes. Can anyone kindly help me choose the best protocol? Any advice?!
Kind regards,
Rashad
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Hi Rashad,
as mainly the more mature stages can be found circulating, a common way of detection is using the Pfs25 QT-NASBA. 
Andrea
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Hi,
I am doing the molecular characterization of Plasmodium species in the area of Kedougou (Senegal) and I would like to have some more information about the MSP-1 gene (chromosome location, allelic diversity).
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Many  thanks Surendra Kumar Prajapati
I think  your answer is quite clear for this question
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Cells are P. falciparum freed from RBC's. This should be a quite fast method since I want to study transport processes.
Thank you.
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In this case, a gentle cell sorting with SdFFF could be a good solution, as the radiolable eluted in the void volume, while cells are retained by they size and density.
Perhaps something such as elutriation could be also interesting for you.
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When should we proceed with splitting the culture?
I am following the protocols of the paper below:
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When should we change the media while culturing Plasmodium falciparum?
Well, I would say that depends on how high the parasitemia is, I normally keep them around 5% and change medium everyday (independent of the stage). If the parasitemia is low (around 1% or below) and they are ring stages is ok to skip media change for one day. 
When should we proceed with splitting the culture?
I normally split when the parasites are in late trophs-schizont stages and the parasitemia is above 1.5% (that also depends on the multiplication rate of the strain being used). In my case the multiplication rate is around 6-8, meaning that if one day they are at 1.5% and I don't split them, next day they will be around 9-12% which is the maximum recommended parasitemia for a continuos culture. If the parasitemia is higher than 1.5% I definitely split them otherwise the culture will crash, I adjust the parasitemia depending on what I'm planning to do with them, if needed for experiment you will need rather high parasitemia but if it is just to keep them up and running you can have low parasitemia and therefore you save effort and m