PCR - Science method

I am using nested PCR to determine the alleles present in my parasite population for a specific gene. This has gone well, but one gene has given me an issue.

The initial PCR for gene MSP1 resulted in a product of 477bp which was expected. The following reaction should produce products between 100-300bp, however, I am seeing those amplicons in some samples as expected but there some samples have a band around 500bp.

The primers are from literature and I have not designed them.

Is it likely the primers are non-specific? In this case where the expected amplicon size for the allele is known do I then ignore these larger amplicons?

Any help would be appreciated

Edited to add: I'm using the PCR conditions specified for the mastermix with the annealing temperature as determined by the supplier's annealing temperature calculator available online.

the primers are going to be used to detect Klebsiella pneumoniae specifically, so I made it from specific sequence after looking on NCBI. The sequence was from Korea, while I tested the primers using samples from Indonesia. The PCR settings I used was pre-denaturation at 95 degrees Celcius for 5 minutes, and then 30 cycles of denaturation at 95 for 1 minute, annealing for 30 seconds, elongation at 72 for 30 seconds, then polymeration at 72 for 10 minutes.

once a friend suggested to try using the same settings, but instead of 30 cycles, we used 40 cycles, took half of the sample's volume for electrophoresis, and I got single band on the right spot but too faint. So we used the remaining volume that is still inside the PCR tube, for another 40 cycles, and I got a single band and very visible.

I ran 3 samples on 3 different degrees (56, 57, and 58 degrees Celcius), and the result at 56 was very visible and on the right spot, at 57 was very visible but below 100bp, at 58 was very visible but a little bit higher than the 56 one.

What does it mean ? Does my target sequence has a very low copy number that it requires a very long PCR time for it to be amplified enough to be detected ?

hii,

I have one group in six different ZTs --> ZT0, ZT4, ZT8, ZT12, ZT16 e ZT20.

I performed gene expression by real time PCR using the 2- deltaCt method

I have used graphpad to represent the curve but I don't know what statistical method to apply to obtain the p value.

Thanks in advance :)

Hello.

I tried to get bands from a few samples using the nested-PCR method, and I tested the 2nd round PCR with 0, 10, 100 and 1000 dilutions of the first product. Only in 0 dilution, smear is seen. Where is the problem with this test and how can I optimize this test?

Hi all, I am struggling to transform my BY4742/1 cells using the Gietz protocol along with a linear PCR fragment. I understand that using a PCR gene disruption cassette is not as efficient as using a plasmid for homologous recombination however this is the situation I find myself in. My fragment is ~ 2kb and I have used a high fidelity pol for amplification. I'm wondering if anyone has any experience in doing a gene deletion this way and how much DNA & ssDNA carrier you used for a successful transformation. Would it possibly be better trying electroporation and if so how much DNA would be suitable in that situation?

Thanks!!

Hi everyone,

I am trying to knock out a non-coding region downstream of my gene of interest. I looked at a publication which did exactly this but in a different species.

They seemed to use two sgRNAs flanking the region of interest and didn't mention any additional components to the knockout in the method section. They show almost complete knockout in their paper (using PCR, the bad after KO is 500bp shorter than in the mock)

My question is; is this a common approach? Just using two sgRNAs a few hundred bps apart without any HDR template and hope that the NHEJ leads to a loss of the region between the two sgRNAs?

Does the DNA remain stable or degrade at this temperature? Would there be any difference in thermal stability between supercoiled and linear forms of say, 3 kb plasmid.

Dear Researchers,

I did stable transformation in passion fruit and now doing detection of mutants, I run the PCRs using the mutants DNA (Target), control plants without target gene DNA ( CK), Agrobacterium/ plasmid (positive), H20 ( negative control) with the target gene primers (around 1700 bo product length), later run the 1.5-2 % Gel, but the bands of mutant samples were above the positive band, mean above than the target product length which is around 1700 bp, as the positive (Agrobacterium showing exact bands) but the samples showing around 2000 bp or above, marker 2KB, I have attached the picture, underlined are all mutant samples, both side 2kb marker, and +ve mean the known and confirmed Agrobacterium with around 1700 bp of the target gene.

so what possible problem and solution about these having bands above the target?

I shall be thankful

Regard

Rizwan

I have been dealing with degenerate primers for paramyxovirus virus detection. The detection assay calls for a heminested PCR with the same reverse primer used for round 1 and round 2 of the PCR reaction. The issue arises when trying to perform round 2 using the round 1 PCR template.

I wanted to ask if there is some specific way to dilute/ purify the round 1 PCR products to get proper bands in round 2.

Also, i did not experience this problem when dealing with Nested PCR primers that are designed differently for the forward and reverse reaction.

I was trying to use Gibson Assembly to fuse 2 PCR products (although it is not really used for this purpose). Upon Gibson reaction of the gel extracted PCR products, I tried to amplify them with Q5. On the test gel, there was only short bands slightly above primer dimers. I was curious if Q5 is faulty and I tried to amplify only one of the PCR products with Q5 (I know for sure that the primers work since PCR from cDNA works and forward primer was successfully used for Sanger sequencing), the result was negative: Q5 did not amplify anything from the gel extracted PCR product. Did anybody experience the same/similar thing?

Hi guys,

Attached herewith is a 1% agarose gel picture of my PCR reaction (triplicates). As my total reaction volume for the triplicates were each 50 ul, 25 ul of the each reaction was loaded into one well. The profile of the triplicates was different. Only one reaction was showing the band of interest (indicated with a red box), while the other two were smeary. This would mean that the PCR reaction is inconsistent. I used 10ng/2ul of cDNA for my reaction, 0.3 uM of the primer pair, 0.3mM of dNTP, 0.5 U of KAPA HIFI DNA polymerase.

My PCR cycling conditions are as follows:

Initial denaturation: 95 celsius for 3 min

Denaturation: 98 celsius for 20 sec

Annealing: I did a 2 step cycling conditions, 52 celsius for 15 sec for 5 cycles, and 62 celsius for 15 sec for 30 cycles.

Extension: As the fragment that I want to amplify is around 2 kb, I used 2:30 min at 72 celsius

Final extension: 72 celsius for 5 min.

I am planning to add in DMSO, but would like to hear from you guys first before I proceed.

Please advise.

Thank you.

As stated, I would like to know what is the difference between NCBI blast and EzBioCloud?

I have sequences of multiple unknown bacteria and am trying to do the first identification on them using only the 16s sequence by sanger sequencing. Once they have been screened, I will pick the one that looks interested to move forward with the characterization.

However, when I tried to use these two services, I have gotten a bit different results.

For example, in NCBI blast, the sample I have generated more than 10 hits that have < 99% identity. However, in EzBioCloud, I can only get one hit up to 98.99% of similarity and it doesn't have a strain name or taxon name. The highest one with strain name and taxon name only has 97.91% similarity. Therefore, I don't know if this one can be considered a potential novel strain or if someone might already have isolated and identity it.

I was wondering what's the difference? Thank you.

Hello,

I am using the Qiagen PCR clean-up and purification kit. I am purifying a PCR product of 750 bp.

I have added the buffer PB as described in the manual. The color of the mixture was violet (too high pH) so I added 10 microliters of 3M Sodium acetate (pH 5.0) as advised by the manual. However, the mixture remains pinkish in color.

is there any other way to regulate the pH and lower it?

I have used 10 microlitre forward and 10 microlitre reverse sequence stock to make 100 microlitre working primer solution is that the reason for these kind of bands what is dimer ?

Dear All

We are trying to detect Wolbachia in Culex and Aedes spp (adult, larvae and pupae). Can anyone please tell what would be the preferred approach to start with considering adult moquito samples. For instance, some articles refer to use to whole body while others have isolated gDNA from organs such as ovaries. It is also mentioned that removal of head improves detection due to presence of inhibitors. It is also proposed somewhere that Wolbachia could exhibit tissue tropism. Kindly share your experience and expert advice on said query.

Looking forward

Thank you

I got a problem in my PCR product sequencing. The agarose gel electrophoresis showed that the band of PCR product is single and strong, though a smear which is smaller than the desired product can be seen. I cut gel and purified the PCR product and send it out for sequencing. However, I got bad sequencing results. Has anyone else run into this issue before? Could you give me some suggestions? Thank you.

Attached are some example file that were giving me trouble.

Hi,

I wonder if anyone knows how to use sample release reagent from Sansure Biotech ?

I electrophoresed 6 ul of PCR product for 90 min at 85 volt in 2.5% gel and TBE buffer. my PCR product length is 490 bp.

I want to do MSP for InsR and slc2a4 gene. My template DNA comes from skeletal muscle and blood of diabetic Mus musculus. After DNA extraction I treat the DNA with bisulfite treatment. I'm still in optimization stage. Before MSP I do nested PCR to my bisulfite treated DNA. I tried to change primers concentration (10mM, 5mM, 2mM, 1mM, 0.5 mM 0.25 mM, 0.1 mM), used temperature gradient for annealing temperature (for both nested and MSP), and adding more template.

I uses PowerPol 2x PCR mix by abclonal as my mastermix. My friends tried to use the same DNA and they got their target band (Their target genes are FTO, KCNJ, PDK4, and PPARG). I set the PCR program exactly the same as the mastermix user manual suggestion. My mix contains 25 microliter PowerPol 2x PCR mix, 1 microliter primers (I have tried 10mM-0.1mM), 1-5 microliter of template(bisulfite treated DNA for nested PCR, Nested PCR product for MSP), and adding Nuclease Free Water until the final volume 50 microliter. What should I do to make my target band appear? The only things that appear in the gel are non-specific bands.

Hello everyone.

I am working on Aspergillus sp., so I ordered primer specific to the Aspergillus genus targeting ITS1 and ITS2 markers. I tried the primer on a positive control and one of the isolates that confirmed by morphology that they are one of the Aspergillus species.

Unfortunately, the band (595-600bp) was seen in the positive control but not in the chosen isolation.

PCR conditions :

94^oC -5min

94^oC-1min

58^oC-1min

72^oC-1min

72^oC-10min

35 cycles

I have done electrophoresis of my PCR product 70 V for 40 min and the bands were moving away from the ladder range what is the correct way to do electrophoresis?

I am trying to use PCR to amplify my samples with barcoded primers for 16s sequencing. I used Phusion Hot start II DNA polymerases kit. My primers are supposed to target the V4 region and amplify the products around 330bp. However, besides the target products, I found some bigger products (about 550 bp) appeared. I haven't came across the issue before and the negative control was fine (which exclude the contamination from water or materials). The PCR protocol I followed is : The 10-20 ng of DNA was used as template in the PCR reaction (50µL), which contained 10µL HF buffer (Thermo Fisher Scientific), 1µL dNTP Mix (10mM; Bioline, London, UK), 1U of Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific), 500 nM of each barcoded primer. PCRs were performed with an Alpha cycler 1 (PCRmax, Staffordshire, UK) using an adaptation of the cycling conditions of Caporaso et al. (2012). The cycling conditions consisted of an initial denaturation at 98◦C for 3min, 25 cycles of: 98◦C for 10 s, 50◦C for 20 s, and 72◦C for 20 s, and a final extension at 72 ◦C for 10min. The size of the PCR products (∼330 bp) was confirmed by agarose gel electrophoresis using 5 µL of the amplification-reaction mixture on a 1% (w/v) agarose gel.

Few thoughts from myself:

1. the DNA template I add was too much, I add about 100 ng DNA per reactions, which might increase the unspecific products. However, my cDNA samples also had the bad as well.

2. the initial denaturation is too short

3. the annealing temperature was too low. the lowest Tm for my primers is 59 degrees.

I got primer dimers for my PCR. Then I tried to use hot start master mix for PCR and ran the gel. I got multiple bands for hot start PCR product. What is the reason for this.

So, I've been doing DNA extractions and PCR of mushroom collections and cultures, and I'm having mixed results. Routinely, we've been diluting all our DNA extractions if it's above 50 ng/ul, but sometimes I'm not having good bands on gel after PCR, and I'm worried that my DNA concentration is not optimal, is there a certified optimal DNA concentration for PCR?

Doing SDM, getting PCR but not getting transformation colonies. What could be the issue? Comp cells are fine when used control.

-PCR: 10 cycles (40ng plasmid template), Can See right size PCR band on the gel.

-PCR digestion with DPN1 (1ul 2hr)

-Heat Inactivation 72℃ for 20 min.

-For transformation 0.5, 1, 5, 10 ul of above mixture in to 30ul of JM109 competent cells Kan+plates. (Control plasmid total 10ng in 1ul volume for 30 ul cells(same) gives colonies)

So no colonies on SDM mixture. What could be the issue?

Dear Community, I extracted DNA from yeasts and performed a PCR with ITS primers. The PCR bands are not intense and do not allow sequencing. I've tried to extract DNA with a lithium protocol, but the bands are still weak. Do you have any recommendations to have better yields of my PCR products?

I have done gel electrophoresis of my PCR product the results were shown like this there were no proper bands observed in the gel and some of the amplified DNA bands were stuck at the well region can anyone tell me what could be the reason for this?

Hi, I'm Siska

I've do PCR product cloning for blunt end PCR Product. It amplified by using KOD FX Neo (Toyobo). But, the product PCR didn't ligate to pTA2 vector. I've try twice by using pTA2 cloning vector, but it's failure. What should I do with this problem?

Thank you

When I measure the concentration and quality of the purified PCR product, the value of 260/280 is within the range of 1.8 - 2.2, while the value of 260/230 is always close to 0. Why is this happening? Can it present any complication for subsequent procedures?, such as performing another PCR from that purified product. And finally, would it affect nucleotide sequencing?

I am using Step One real-time instrument and the Green master mix with ROX.

Thank you all

Hi,

I want to PCR a 200nts sequence from DNA samples that are extracted from fixed cells. For DNA extraction, first cells were lysed in lysis buffer overnight along with proteinase K treatment followed by DNA separation using phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitation. DNA yield and quality were similar to unfixed samples, which was satisfying, but there are no PCR products using DNA from fixed cells. Do you have any suggestions on how to prepare a fixed DNA sample for PCR? I will test whether fixing with 1.5% PFA resolves the PCR problem. Do you think using FFPE DNA extraction kits could help?

Thanks

I have recently obtained a mouse model B6;129-OoepGt(139A2-3)Cmhd/Mmucd from MMRRC

I am having trouble getting our genotyping protocol to work and would like to find the sequence of the gene trap insertion in context of the mouse genome. In other words I want the sequence of the gene including the insertion.

There are elements which could be targeted for PCR such as the LacZ or Neomycin cassettes however from an academic viewpoint this sequence should be available no?

I have found the sequence of the inserted plasmid Gep-SD5 but I would just like to explain differences in PCR amplicons from what we expect. There are a bunch of links on the MMRRC page but I find myself lost.

Any help or pointers would be greatly appreciated!

-Nick

I induced two cultures at 0.5 OD600: 1 with just the vector, and another with my ORF in the vector. However, following induction with 1mM IPTG with shaking at 140 rpm at 37 degree C for even 6 hours did not induce protein overexpression, even in the culture harboring just the vector. The vector and the clone are both proper (as confirmed by sequencing, digestion profile, PCR screening).

Help and suggestions would be appreciated.

I have GeNei PCR Master Mix of 2x concentration shall I dilute it to 1x before using for PCR or use it directly without any dilution

Can someone tell me what is the best way to use Master Mix

Hi, i'm trying to cut the SARS-COV2 S protein from the plasmid pGBW-m4046887 to get een empty vector with small 6-His tag protein which I want to use for negative selection for SELEX.

I did PCR and now i want to make PCR clean up, then phosphorylate the ends and then ligate.

Could anyone correct me if my plan is wrong:

1. After PCR clean up meten de concetration of dna

2. Phosphorylate with T4 PNK in T4 ligase buffer:

100 ng dna

1 ul T4 PNK

2 ul T4 ligase buffer (10X)

milli Q water until 20 ul

30 min incubation , 37 gr. Celsius

20 min 65 gr. Celsius

+ add 1 ul ligase and incubate 2 hours at room temperature.

I'm not sure if could just add 100 ng dna, because i'm not quite sure how to calcultate the right amount.

The plan is to transform this vector to Neb-10Beta celles, isolate the vector and sequencing.

Hello!

I have black C57 DsRed & EGFP mice (from JAX) and need to quantify the expression of EGFP or DsRed at mRNA levels with qPCR. I am wondering if anyone knows about the primers?

Thanks !

P.S

I have the primers for the genotyping of these mice but I think those primers are too big for this purpouse.

Dears researchers,

Has anyone had the error below when calibrating the Step One Plus equipment (Thermo Fisher Scientific)?

"Spatial Calibration failed: Well locations are not evenly spaced.

System will revert to previous calibration.

Exit the calibration wizard and refer to the Hrlp to troubleshoot the calibration failure.

Error Code 1302"

I can't find the error code in the troubleshoot. Company support has not found a solution yet.

I already did the decontamination and the Backgroud calibration worked.

Regards,

I'm doing 16s rRNA amplification using DNA extracted via the boiling method for several bacterial samples.

The quantities of each PCR reaction are;

DNA template - 1 ul

Forward primer - 1 ul

Reverse primer - 1 ul

Nuclease free water - 9.5 ul

Go-Taq Green master mix - 12.5 ul

5 ul of each PCR reaction is loaded into the 1.5% agarose gel.

What could be causing this?

I am trying to amplify ssDNA product using PCR. I am just wondering whether following the normal PCR protocol will yield a product since the starting DNA is not dsDNA. Have anyone tried amplifying a ssDNA or know a protocol that I should follow?

I bought a synthetic gene from a vendor, the gene is ligated in the pUC57 plasmid (with ampicilin resistance). the pUC57 plasmid have been tested using electrophoresis and it's all good.

I'm trying to transform it to the DH5 cells to store and propagate the plasmid, but it's failed (it's been 6 months trying). i have been using some methods such as CaCl2 and SMOBIO CK1000 transformation kit, heatshock. and using ampicillin LB plate (100 ug/ml and recently i use 200 ug/ml)

i have specific primer to detect the gene inside pUC57 plasmid, the colony i pick is shows positive result (PCR) but when i isolate the plasmid (from liquid culture using GeneJet Plasmid MiniPrep kit) and run it in agarose gel electrophoresis it shows smear (really thin) i also run it with positive control (also DH5 that have a known plasmid in it, so i think this could ommit the possibility of dnase contamination in the plasmid isolation kit).

does anyone have ever encountered something like this? or anyone have any advice? thankyou in advance:)

Hello,

I have a significant difference of the housekeeping genes between the control group and the rest of the groups in my PCR results. The reason for this is that I had to transcribe the control group individually (RNA -> DNA). Can I still evaluate the control group after normalisation or do I have to restart the experiments?

Many thanks!

Hi,

I did this PCR using Platinum™ SuperFi II DNA Polymerase following the instructions in the protocol.

I tried multiple conditions (gradient PCR, longer extension time, less amount of DNA etc)

My expected fragment was around 250bp, and I did get it, however there is a double band that I have not seen before.

The band can be observed in all samples and I'm not sure if these are primer dimers or something else.

I've used the KOD polymerase before with the same primers and I haven't had these issues.

Any ideas?

I am looking for to buy a RT-PCR machine-96 wells. Please mention your experience with the machine in your lab. Any pros and cons. Thanks

Hello

I used 2% agarose gel 1X TBE buffer (pH ~9) for gel electrophoresis. For electricity supply I used 60V and 300A for 3m min. I used my known PCR product in this gel. After 10-12 min I found my PCR product in the gel. However after 30 min my product was vanished from the gel. I also attached my gel picture for your reference.

I have designed a primer set for LAMP and couldn't make it work. I am using NEB WarmStart® Colorimetric LAMP 2X Master Mix and tried different types of samples. Only thing I am worrying about is that my inner primers don't work. I know F3 and B3 works fine because I checked them in Real-Time PCR machine.

Also attached an image of gel electrophoresis of 3 positive and 3 negative control sample.

I would appreciate any help.

I am not getting any amplification for my target gene in real time PCR. The cDNA samples are fine since the amplification in positive control is fine. I have also obtained amplification for my target gene in semiquantitative PCR using annealing temperatures 60 and 62. Does any one has any idea where the problem is.

I would like you to help me solve some doubts about the PCR conditions to obtain good quality products for subsequent sequencing. I am going to perform PCR targeting 16S, and I want to know if there is a range in the concentration of universal primers that works best (I usually use 0.4 uM of each primer) as well as a concentration of DNA to add in the reaction (I usually use 100 µM). ng) and finally if the appearance of the PCR product in the gel indicates that it is optimal for sequencing. So far I have obtained chromatogram results that are not very good, with overlapping peaks or longer sequence lengths than expected (extremes with low peaks). If anyone has suggestions I would appreciate them.

Sorry for my English

I want to add dATP on the ends of to my PCR product (Phusion, Thermo). My question is if it's possible with use of DreamTaq polymerase (Thermo)? I find informations about Taq polymerases but can not find out if i can use this polymerase to do that (a-tailing, not PCR itself, i got insert from PCR with Phusion polymerase).

Thx for help, cheers

I have extracted DNA from fecal sample using salting out method.The concentration under nanodrop comes more than 100 and the ratio A260/A280 ranges between 1.5 to 1.9 whereas A260(10mm) is morethan 5. The gel image has been attached here also. Can somebody explain what could be the possible reason? (Note: Primers, PCR master mix, PCR conditions are fine using control samples)

Instead I am getting a smear with a brighter illumination near the well.

In my experiment, I use a plasmid (~16kb). What I want to do is to delete Luciferase gene from that plasmid by using a specific pair of primers with PCR technique. Last time, I tried and I got no band at all. So I wonder that if it is possible to extend the whole plasmid except Luciferase gene by PCR as the plasmid size is pretty big. Thank you.

Dear all,

I am planning to do in vitro transcription from a PCR template. However, I need a poly(A) tailed RNA product. If I add 30 nucleotide poly(A) tail before the in vitro transcription, would it stop the T7 polymerase reaction earlier? Thanks

Cheers,

I'm trying to look for a specific field which people makes fungi molds to identify the species. I'm planning to make it as a machine learning project. Like in the field of agriculture, medicine. Thank you

Rt-pcr related question

I want to expose my lung epithelial cells to different treatments in vitro and then detect pro- and anti-inflammatory cytokines/chemokines in the conditioned media - can I then do PCRs on the conditioned media? Any guidance would be very much appreciated!

Real time PCR related question

Hello I need help putting together annealing buffer. I was given the following:

H2O supplemented with 10 mM Tris-hCl pH 8.5 and 50 mM NaCl

We currently have 10mM Tris-HCL, nothing higher. Therefore I am worried that when I put together the buffer, the diluted Tris-HCL will affect annealing.

Would my annealing reaction still be okay and should I adjust the concentration of NaCl?

I designed the primers by myself (with a Tm of 57.3 and perfect binding to the template, checked for no secondary structure) and the expected amplicon size should be around 7.0kb, but I could not get a 7.0Kb product at the end of each PCR, instead it was a band that did not seem to look like a PCR amplification.

The ladder used was a 1KB ladder. The DNA in the sample was extracted and tested by Nanodrop and the A260/A280 was 1.92, giving a DNA concentration of 1.3ng/uL. I referred to the paltium superFi ii handbook and used a 20ul PCR protocol (10ul mastermix; 1ul of 0.2um forward primer and reverse primer; 4ul of DNA template; 4ul water).

PCR run times were also referenced from the handbook recommendations. But every time the amplification came out strange and I set Negative control (no sample added) and still got the same result.

The attached pictures are my electrophoresis results, well1 and well8 are 1KB ladder. well 2 is undiluted sample（4ul——1.3ng/ul DNA concentration）, well3 - well6 are PCR for samples diluted 10, 100, 1000, 10000 times respectively. well7 is negative negative control (no DNA template added). Please give me some suggestions, I have tried many times with this result

Hi there, thank you all in advance for your help!

I'm having an issue with my bands being ~500bp too short in the second round of nested PCR.

Here's my protocol

First round of PCR

-amplicon is ~2.2kb

-40cycles of PCR

-I get very clean bands with when there is sufficient template DNA. For samples without extremely low levels of template DNA, I move onto nested PCR

second round

-amplicon is ~1.8kb

-30 cycles of PCR

Explanation of gel below

Lane 2 of the gel below is the expected length of the amplicon for these primers. In this well, I use my positive control template DNA that did NOT undergo a first round of PCR. lanes 3-11 all are a 1/10 dilution from the previous PCR reaction. Lane 3 is the positive control from the first reaction.

My problem/question

For lanes 3-11, the band is about 500bp too short. I can't figure out why... Your help is greatly appreciated

Hi all,

1. How do you identify if you have bubble library or just insufficient size selection?

2. How to deal with bubble library? Would reconditioning PCR work? Is it recommended to use single primer or both forward and reverse primers?

3. How to quantify bubble library for library pooling? qPCR? How?

Thanks.

Lux

I’ve been extracting very low copies of viral DNA and RNA and performing a PCR afterwards to detect what I extracted. When I run PCRs for viral DNA, the PCR always works and my negative control is always negative. But for viral RNA (converted to cDNA), the negative control shows a band when I run an agarose gel. I fixed this problem a few times in the past by changing primers but now if I change everything the negative control still has a band.

The negative control band is the same size I expect from my samples meaning that there’s template in my negative control. I also ran a qPCR and the melting peak in the negative control is also what I expect from my samples.

I’ve tried changing the polymerase, water, primers, lab coat and using 70% ethanol but I can’t seem to find the source of contamination. I’ve changed my pipettes, tubes and bench but that didn’t work. I ran another PCR without any positive controls (to avoid cross-contamination) but my negative controls still show a band.

Any suggestions on how to fix the problem would be appreciated. Thanks.

Dears, I am doing qpcr of genes with cdna made of RNA from adipose tissue. In the efficiency curve of the primers, the cdna serial dilution does not promote adequate amplification. The less diluted points dots are having less amplification. Example: in the photo, the last amplification is the highest dilution (1:2). I believe it is the presence of an inhibitor. Has anyone dealt with this? Know what to do? The 260/280 and 260/230 ratios are fine. The profile on the electrofluoresis gel is also good. The RNA was extracted with silica glass beads (sterilized and washed with acid), Trizol according to the manufacturer, and then the material was washed with precipitation with 3M sodium acetate and 100% ethanol. Resuspended in nuclease-free milliQ water.

I have one PCR workstation.

I performed my RNA extraction/cDNA synthesis on a separate bench (outside hood).

I now have very concentrated cDNA.

In previous labs I had 3 work stations.

1: Prepare primer/probe working stock

2: Prepare serial dilution of concentrated template

3: Preparing master mix and plating serial dilutions

I now have one workstation.

Would you perform you cDNA dilution outside of the workstation?

Or would you do it inside and just turn on UV light/spray DNAzap before plating?

Any advice on PCR workflow would be useful.

Thank you!

Hi, I'm trying to make random mutagenesis on my interest gene. I want to use error-prone PCR. But I only know how to amplify and set condition for best mutagenesis rate. I wonder how can I ligate these products after amplification to my desired plasmid? Can I use infusion or just ligation by T4 ligase?

In the last few days it was not possible to access Miner website. Does anybody knows what is happen with the website?

Thank you!

Camila Carvalho

Why hybridization based targeted exome sequencing library comes very high concentration that is 53ng/uL (expectation 1-5 ng/uL).

No change in the PCR cycles and master mix

No change in Beads concentration during clean up step.

Also no change in elution volume.

Your kind answers are highly appreciated.

I have ran and reran this PCR so many times and am at a loss for what to do at this point. I started by cutting off a small piece of ear (about the size of rice) using a razor blade and tweezers. I used 70% ethanol spray to sterilize by tools between ears.

I then performed an extraction using chelex beads. 5g of beads in 50mL of nuclease free H2O. I used 100 uL of beads per sample. I used a shaking heater block set at 95C and heated the samples for 40 minutes vortexing every 10 minutes. I then centrifuged the samples for 2 min at 20G. I pipetted off the supernatant leaving the beads and the remaining ear behind.

I then ran a PCR. I used 1x PCR buffer, 1.5mM MgCl2, 0.3 mM DNTP, 0.25 primers, 1U taq, and 1 uL of DNA.

That extraction method didn't seem to be working so I used Platinum Direct PCR Master Mix to extract DNA from the ears. I cut a small 1cm piece of ear and added 20uL of lysis buffer and 0.6uL of Protinease K to each ear. As per the instructions I heated the samples at 98C for 1 min then centrifuged them at 20G for one minute. I pulled off the supernatant and used 2 uL to run a PCR. That seemed to work but I saw lots of high MW products so I centrifuged again at 20G for 20 minutes and ran the PCR again. My bands dissapeared.

So I decided maybe I needed to lyse them for longer. So I repeated the extraction but heated for 30 minutes and centrifuged for 10 minutes. This didn't work and it looked like the DNA was trapped in the wells of the gel. The guide for the kit suggested adding 1uL of Protinease K post PCR. So I used the same DNA and ran another PCR but added protinease K following PCR. This didn't work either.

I am not sure what to do at this point. I have attached the PPT that has all the gels I've run and the conditions.

I am seeing multiple bands in my PCR reaction. Is it possible that my stock is contaminated or issue with a gel run?

Direct PCR lysis buffer is one of the fastest methods to do genotyping. By taking a piece of tissue and placing it in a small volume of Direct PCR lysis buffer you can then take a few microliters and do a PCR directly. I would like to do a similar approach but performing a restriction enzyme digestion instead of a PCR. The issue is that the DNA is in a buffer of unknown composition and I am not sure whether this will affect the restriction enzyme reaction. Have anyone experience with that? Thanks! Miguel

Hello everyone

Thank you in advance for your help

My Melting curves in qpcr look like this and have among other plates as well little peaks after the first one. I am not sure what could cause this as Ive ruled out most technical issues. Could it just be due to operator errors or what might cause this. I will drop some examples below so any help would be appreciated thanks.