Science method
PCR - Science method
PCR is an in vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.
Questions related to PCR
Hi everyone,
I was running a PCR and, unfortunately, the thermocycler died around the 7th cycle. It felt like a waste to simply throw away the PCR, so I put it on another thermocycler and lowered the cycle number to 27 (it is usually 34). Has anyone had experience with changing thermocyclers during PCR and what were your results? Should I even use this PCR for downstream steps or should I just re-do it entirely?
The thermocycler settings are:
1) 94C, 3 min
2) 94C, 30 sec
3) 55C, 30 sec
4) 72C, 45 sec
5) Go back to Step 2, 34x (again, I lowered this to 27 when I changed thermocyclers)
6) 72C, 20 min
7) Hold on 4C
Thank you.
Hello. I'm looking for suggestions on troubleshooting index (or barcode) PCR. Specifically, my problem is that some of my sample DNA concentrations dropped after adding the indices/barcodes. My ideal concentration is no lower than 9nM. One 96-well plate had 5 samples and another plate had 28 samples that were below 9nM.
For context, I'm adding Illumina indices to my mixed amplicon (combined ITS and 16S) sequences. After the index PCR, I cleaned up my samples and quantified them with Qubit. I had some samples that dropped significantly in their DNA concentration after this step (index PCR). For example, one sample had 133nM (measured after 16S amplicon PCR), but then dropped to 8nM (measured after index PCR).
Prior to indexing, I diluted the separate ITS and 16S plates to 20nm then combine 2uL of each of the 20nM diluted plates together. From the now mixed 4uL, I then use 1.5uL of each mixed amplicon sample for index PCR. For the index PCR, I typically use Kapa HotStart ReadyMix (7.5uL for 1 reaction), water (3uL for 1 reaction), and 1.5uL of each index (for each sample).
The thermocycler settings are:
- 95C, 3min
- 95C, 30sec
- 55C, 30 sec
- 72C, 30sec
- Go to Step 2, 7 times
- 72C, 5 min
- Hold on 4C
I've already increased the DNA concentration input for the index PCR. Originally, it was 10nM, but 20nM worked better. I'm unsure about increasing the DNA concentration again as many of the samples lowest concentrations (prior to indexing) were around 20nM.
Another suggestion was to toggle with the thermocycler settings, but I'm unsure how to optimize these as other samples worked fine with these exact settings.
Thanks for reading this very long post. I'm open to any suggestions on how people have troubleshooted indexing!
Vectors which can accept PCR products up 700-800bp
I ordered a primer when I received it where one nucleotide is different in the forward primer.
the original sequence is: CTCTTTGGGCTCAGAGTGAGTCTGG
the sequence which I get: CTCTTTGGGTTCAGTGTGAGTCTTG
Three nucleotides (Bold) are different
This primer will work?
while I have tried so many times but there is no result. If it is working, how can I fix my PCR protocol?
What do you do when you have tried everything to transform your yeast strain and you see a lot of colonies but most of them are false positives.
Regarding confirmation of transformation, I isolate the gDNA and then perform a PCR but for some reason I do not see any bands even with the positive control. My technique is fine and I tried every possible change with no results. HELP ME!
I have been doing PCR genotyping for mouse samples for 2 years. It worked well before, until I ordered new gotaq polymerase last month. I kept the polymerase at -20C. When I used the new polymerase, I did not see any band, but when I increased the amount of taq pol, I could see the band. I then used it again for current genotyping, but it did not showed any band, even when I increased the taq amount per reaction. I used positive and negative control that previously worked, but they also did not showed up. I tried changing the taq, PCR water, dNTPs, and diluted fresh primers from 100uM stock, also dilute DNA and increased PCR cycle. All cannot solve the problem. Then I bought new primers and collected new tail, and suddenly it worked and showing band nicely. However, when I repeated the experiment (only one day apart; using the exactly same reagents), I could not see any bands.
Can anyone help me with this issue?
i have been struggling with this for 3 weeks.
Thank you!
Hello
I am having a trouble with the result of agarose gel electrophoresis of the PCR product of Entamoeba gingivalis
DNA
Samples:
Paper point samples from the sulcus(between tooth & gingiva)
Stored in a PBS transition solution
DNA extraction:
1-samples heated to 37˚for 10 mins and mixed well on vortex.
2- 0.3ml of the suspension was washed 3 times with distilled water and spin down between each wash at 14000 rpm for 3 minutes
3- the pellets were re-suspended in 100 microlitre nuclease free water.
Pcr procedure:
1- Primers forward and reverse specific for E.gingivalis were used
2- GoTaq green master mix was used
3- The master mix consisted of the following:
A- 150 µl master mix(GoTaq green)
B- 6 µl forward primer
C- 6 µl forward primer
D- 114 µl nuclease free water
Divided on 12 samples =23 µl
Add 2 µl of each specimen to be the total of the mix 25 µl
4- The reaction cycle was prepared as following:
A-Initial denaturation 95˚ 2minutes
Then 30 cycles of the following
B- 95˚ 30 second
C- 55˚ 30 second
E- 72˚ 35 seconds
Final extension 72˚ for 5 minutes
Question is the following image of the electrophoresis is typica
Thanks in advance
update:
the expected size of the PCR product is
135
in the previous image
today i changed the voltage to 80
and the running time to 40 minutes
and got the following result.
Named IMG_3106
the first 10 lanes are the pcr products
11th is the ladder
20th negative control
update:
20 oct 2024
thanks alot for all the significat paticipation
the agarose gel is prepared by adding 1.5 gram powder to 100ml of TBE (1X) and 5µl of gelred
attached a new picture named IMG_3249
is a photo of the electrophoresis
of a positive control by doing
pcr for RNA 16 s
thank you
the last row is the negative control
When I used 95bp PCR product (full-length tRNA with promoter), the T7 polymerase RNA synthesis kit (NEB) gave a lot of large bands (more than 200bp). Can you explain the reason and the way to remove all the bigger RNA bands.
Hello,
I am using amaR one PCR mix. My anticipated amplicon size is about 500 bp. I need help understanding the darker band at the bottom of each well. Is it the primer dimer or the Amaranth stain that comes with the PCR master mix?
Please help.
Thank you
Hello!
I am optimizing conditions of stem-loop RT-PCR to compare the expression of miRNAs in different cell lines.
Now I'm performing the reaction in one step: RT with stem-loop primer and subsequent real-time PCR with a pair of primers and SYBR Green - all in one reaction. So my reaction mix contains three types of primers, heat-inactivated reverse transcriptase, hot-start Taq polymerase, SYBR and all the nessesary components such as ddNTPs and Mg2+.
I use synthetic miRNA and total RNA from human cells as matrices.
When working with synthetic miRNA, everything goes well (Figures 1 and 2), amplification plot is ok and there is a single peak in the melting curve.
But with total RNA I observe a decline of fluorescence in the plateu phase of amplification (Figure 3) and 2 product peak in the melting curve (Figure 4).
Reference gene amplification plot doesn't have such issues.
What are the reasons of the decline of fluorescence after the 30th cycle?
What are the possible variants of the second product here? Primer-dimers observed in no-template control have Tm = 80 degrees C.
Hi, I am doing qPCR experiments. When the reaction finished, I obtain the Ct value of my samples is zero, but they have a peak with my expected Tm. Furthermore, I run the gel electrophoresis, the result also shows that these samples appear a band at my expected target size. But why the Ct value is zero?
During cDNA synthesis, same amount (400 ng) of RNA was added for each sample. The volumes varied for each sample. With the synthesized cDNA we performed conventional PCR for beta- actin. When the gel was run we saw the sample in which the initial RNA volume (not conc.) was more than others had a strong band density as compared to the one in which less volume was used to synthesize cDNA. This has happened multiple times.
Can anybody tell what can be the reason?
The image is of PCR product (RNA>cDNA>PCR) plotted as:
Well 1: ladder
Well 2: PCR product with RNA volume 13.07ul
Well 3: PCR product with RNA volume 2.86ul
Well 4: PCR product with RNA volume 3.86ul
Ask a question for the JAX Stat3/flow mouse and the primers (19436/19437) which was provided by JAX. The expected results of standard PCR showed that WT=146-bp and mutant=187-bp. From the gene blast, the biding sites in the 7 Intron region, how to explain that PCR can extended to 18 Extron to 20 Extron?
Could you kindly guide me through the steps to design primers for the "OneStep PLUS qMethyl™ PCR Kit" to study the methylation status of specific genes?
I have run colony PCR. I was supposed to get amplicon of 300 bp size. However, after running the PCR, i am not getting 300 bp amplicon. The bands are just stuck in the well. What should I do to get exact sized amplicon after PCR?
I am conducting DNA extraction from the juvenile leaves of cherry tomato (F4 generation) for molecular studies. I would appreciate suggestions on the best protocols or methods that provide high-quality DNA suitable for downstream applications like PCR. I am particularly interested in methods that work well with plants that have high secondary metabolites or are prone to contamination. Any specific tips on handling these issues would be helpful!
Thank you in advance for your insights!
I'm trying to clone some large pieces of cyanobactrial gDNA (sometimes even more of them) into a plasmid via fusion PCR. Then, I transform it into E. coli TOP10, as my PI says it works better (for HiFi DNA assembly).
However, I quite high percentage of my positive clones had some problems in the plasmid elsewhere (like missing I think 2.3 kbp of the plasmid; or some mess in another case etc.).
It is true, that when I compared TOP10 and DH5alpha, TOP10 had many more colonies (more than 10-times). However, I think that DH5alpha should be less prone to DNA alterations, right? So should I use rather DH5alpha and hope that I will get at least few colonies, but they will be positive?
I am using PCR product as my template for in vitro transcription and then loading the Rna samples on 8M urea gel. I am observing multiple bands of both low and high molecular weight(my intended product is of 550bp). What could be the reason. Any suggestions?
Hello ResearchGate group,
As M.D.-Ph.D. scientist working on molecular biology, I am wondering if anyone is interested in joining efforts for creating PCR standardization statistical tools.
Best regards,
Alexios
I have synthesized a gene specific primer for amplification of my target gene. However, I am getting amplicon of only 100 bp rather than 210 bp (target amplicon size). So far I have tested so many optimization conditions for it but still getting non-specific band. I have cross checked the primer pair for non specific binding in my extracted DNA, it shows no complementary with any other region except for my target region.
Could DNA from relatives allow polymerase chain reaction (PCR)s to replicate offspring for the deceased? How exactly?
Research Proposal Honor Kirk Aanes
Since we work mostly with genetic models, we usually genotype our mice after ear punching them for further breeding purposes.
Recently our breeding has taken up qutie a bit resulting in a high number of samples and quite a bit of time "wasted" on something that doesn´t lead to "valuable" results but is, nonetheless, essential for following experiments and breeding.
Does any of you know of a way to facilitate and speed-up genotyping? Someting like a machine that performs the entire workflow?
Our current lysis-PCR for 4 different genes-gel loading-electrophoresis and imaging as well as annotation takes well over half a day to a day for 100 samples and is simply no longer efficient to perform.
Thank you in advance!
Details of the experiment:
We aim to clone 3 gene segments into a vector.
Details of the gene segments:
- The first segment has a concentration of 34 ng/µL and is 1445 base pairs long.
- The second segment has a concentration of 30 ng/µL and is 125 base pairs long.
- The third segment, is 4758 base pairs long, with a concentration of 150 ng/µL, and is a PCR product.
These segments were designed to have an overlap of approximately 30 nucleotides with each other.
The vector has a concentration of 29 ng/µL and is 1850 base pairs long.
I have repeated this reaction using various volumes, but sometimes I did not observe any colonies, and other times, when I sent the colonies for Sanger sequencing, I found that one fragment had been inserted while the other two were not, resulting in different combinations.
In the last reaction, I used the volumes recommended by the NEBuilder site:
- Vector: 4.5 µL
- First fragment: 3 µL
- Second fragment: 0.3 µL
- Third fragment: 2.2 µL
- NEBuilder HiFi DNA Assembly Master Mix: 10 µL
- Each are = 0.114 pmoles
I incubated the reaction at 50°C for 5 hours, then performed a chemical transformation into E. coli Stbl3 strain.
I only observed 2 colonies, but after performing enzyme treatment and sending sanger sequencing, I saw that the desired segments were not inserted.
Could you help me understand what the problem might be?
I am a new person, want to understand the process of learning PCR. It is best to be simple and easy to understand with pictures. Among them, I want to first understand the process of standard PCR.
I use gradient PCR for single target with two different Primers set. both the set failed to get the band. 1st Set Tm is 62.9 and 2nd Set Tm is 63.2. anneling Temp. is as follows 55,57,59,62C 1st set give 76bp amplicon and 2nd set will give 147 bp amplicon
I have treated the cells with ligands with different doses and performed PCR to determine cell expression. Now I need to decide which dose I should include for my studies using EC50. Can anyone guide me, on how this should be done?
Where I work, there are three separate rooms for setting up PCR reactions. In room number 2, whener we use UV light for decontamination, there is a particular odor that remains for minutes after opening the door. I have been reading about this, and, apparently, it is ozone. Has anyone here experienced the same? Do you have any thoughts on why does this happen only in one of the PCR rooms? Is it harmful to be in the room smelling this? How can I overcome this issue? Thank you very much!!
I am currently optimizing a RPA-based protocol. As everyone knows, this nucleic acid amplification technique is based on isothermal amplification (around 37-42˚C) in combination with recombinases and single-stranded DNA binding (SSB) proteins.
I have designed several candidate primers to optimize my RPA. All of them were searched in literature and designed considering the criteria to be used in an RPA reaction.
These same primers were verified by conventional PCR (At different Tm: 55-65ºC), obtaining a successful result.
The problem started when I used the RPA master mix (lyo version from Twistdx) and performed the RPA reaction with the same primers and samples used in the conventional PCR. ALL THE RESULTS BECAME NEGATIVE?!
Primer concentrations used in RPA reaction was 400nm (recommended by twistdx) and the reaction time was 30 minutes at 39 °C (conditions recommended for the set of primers tested). Visualization of the amplification was done on agarose gel (1.5%) and doing a posterior melting curve assay. In all cases, no amplification was detected.
Does any one have a clue on what is happening???
I don´t know which other variables I can change to obtain good results
Suggestions?
Thank you
I am trying to run a PCR to verify insertion of my construct into the AAVS1 locus in iPSC using CRISPR.
I designed three primer pairs to amplify the left and right insertion regions (one binding inside the insert, one binding outside; see image), and one primer pair to amplify a region inside the insert. The insert contains a fluorescent protein, which I can see expressed in the cells under the microscope, so I am pretty sure that the insertion has worked correctly; however, I cannot get any specific PCR product for sequencing. (Even if it has been inserted unspecifically somewhere in the genome, since I am seeing the fluorescent reporter in the cells, I would expect at least to get a positive result for the "internal" region.)
I used DNAzol to purifiy gDNA from the cells, and when I checked it on a gel I noticed two additional bands, which I thought might be rRNA (see image), and when I treated the samples with RNAse the bands disappeared, so I continued happily with the PCR.
For the "internal" region, I am able to use the plasmid as a control, and here I can see a specific PCR product with the expected size, however, for all other plasmid / gDNA template combinations, I get a huge amount of large-sized unspecific PCR products (see second image). Which is why I am currently suspecting that something is not right with the gDNA? But it looks pretty good on a gel.
I am using KOD1 polymerase (KOD1 master mix) according to manufacturer instructions when it comes to amplification from gDNA:
PCR: Total 25 µl per reaction
1.25 µl DMSO
1 µl primer fwd [10 µM]
1 µl primer rev [10 µM]
8.25 µl H2O
12.5 µl KOD master mix
0.5 µl DNA (= 25ng)
Init. Denat. 94°C 1.5 min
Denat. 94°C 5 sec
Anneal 58°C 5 sec
Extension 68°C 1 sec
and also tried
Init. Denat. 94°C 3 min
Denat. 94°C 45 sec
Anneal 58°C 45 sec
Extension 68°C 1 min
I have triple-checked the specificity of the primers, and compared with other primers used in literature for the same purpose (AAVS1 locus). I have re-designed new primers that bind in slighly different places. I have tried different elongation / annealing times and temperatures... It always looks the same (large-size unspecific products).
I a last-ditch effort, I cut out pieces of gel from the "unspecific" results around the size where I would expect the PCR product, and repeated the PCR with those as a template - I got some promising looking results on a gel, but when I sent them for sequencing, it was all unspecific.
I am currently at my wit's end and hope someone else has seen something similar and was able to solve it in the end!!
(Plan B will be to re-do the DNA extraction and try again from the beginning I guess...)
PCR is a technique used to amplify specific DNA sequences, and the quality of the DNA template plays a significant role in the efficiency and accuracy of this process.
How DNA purity impacts PCR and what can be done to improve it?
Dinoflagellates often incorporate 5-hmU into their genome; does this modification cause issues with PCR, such that we'd need to use uracil-incorporating polymerases, or is a normal high fidelity polymerase sufficient?
The bands of my PCR amplicons are mostly wonky, and I can't figure out why. When I did the non-touchdown version, the bands were straight, but results were stochastic (i.e. some samples amplified, some didn't). The chromatogram of the sequences also showed a lot of multiple peaks per position. I shifted to doing touchdown to lessen the chances of non-specific amplification. Touchdown seems to work well in terms of amplification success, but the amplicons are wonky on the gel.
I would appreciate any and all insights and advice on how this happens and how to correct it.
Below are my PCR parameters.
Master mix components (for 20ul reaction volume):
2.0ul 10x PCRx enhancer
2.0ul 10x PCRx buffer
0.6ul 50mM MgSO4
0.4ul 10uM dNTPs
1.0ul 10uM H3F/R primer
0.5ul 5U/ul Taq polymerase
1.0ul template (1:1000)
12.5ul ddH2O
PCR cycling parameters (touchdown): 95C/3min; 11 cycles of: 95C/30s, 65C/30s (-1C/cycle), 72C/1min; 25 cycles of: 95C/30s, 55C/30s, 72C/1min, 72C/15min
Gel electrophoresis: 1.0% agarose in 1x TBE, 1ul amplicon, 1ul 6x GLB + SYBR gold, 2ul Hyperladder 1kb, 120V for 20mins
Hello Dear,
Im PhD student
I was using Gibsson assembly, and I cloned my gene onto the Psl18 plasmid . The plasmid was right when I sent it to Sequencing after geting a positives colonies.
However, when attempting to perform PCR and amplify my gene using my glyceral bacterial stock, I obtain a bande that is appropriately sized, but another bande that is nearly the same size as my gene (see gel picture below).
I don't understand why, as I should only have one bande! Can my plasmid exist in two different forms in the Glycrio Stokc 9 (one that is correct and the other that is not) or is there another explanation?
Or should I do the cloning all over again ?
It`s shloud be a primers issues ! (Althought I checked it in snapgene but it`s bande just in my gene of interest)
Also, I test 2 taq (polymerase and Q5 HF) but I got the same results.
I appreciate your help in advance.
Thanks
Ayoub
Some context first:
on all my experiment I used a control sample labeled with PET. I have very little amount of this control sample so i used both primers for PCR PET-labeled. I thought that with both primers being labeled i could use less PCR product and make it last more.
In every FLA i mixed together this control sample with other samples labeled with VIC, FAM, NED so i could compare these three samples against my control. Then i noticed that my control sample has 2 populations. Like if my primers were amplifying two different targets. But we are very sure that this is not the case.
In previous experiments I have used the same primer sequences. but this is the first time I use it with both primers (forward and reverse) labeled. So this issue only appeared when using both primers labeled.
In the last validation experiment i did the following test:
- Amplify using forward pet-labeled primer
- Amplify using reverse pet- labeled primer
- Amplify using forward and reverse pet-labeled primers
- Amplify using forward vic-labeled primer
- Amplify using forward vic-labeled primer and reverse pet-labeled primer
so you can observe that when ever i used a reverse labeled primer the two populations appears.
technical details :
the target region contains tandem repeats, therefore the template has strong secondary structure. GC% is over 65%, PCR reaction was done with taq polymerase with 5% DMSO.
I have a PCR product that I've been using as a component that I'm ligating into a vector. Did sequencing it was confirmed to be correct and did a ligation it worked perfect and was sequence verified. I did two more ligations using the same PCR product into different vectors and now there is a mutation in the PCR product? How does this happen? I've essentially sequenced it twice(one time just the PCR product and then again to make sure the it was ligated correctly) and it was fine and now the sequencing is showing a deletion. I send to two different companies for sequencing and they have both come back with the same results each time. The first 2 times both showed it to be correct the last 2 times there is a mutation that both companies are picking up on.
When we perform statistical analysis on qPCR data, do we use fold change or ΔCt?
These two words are similar in the meaning of tech.or there is some different between them
I have been working on a knockout in P. aeruginosa, however, after the final PCR check, I see both wild-type and mutant bands. Any suggestions?
Hello, I am currently learning to generate Agrobacterium deletional mutants by homologous recombination. I already prepared the construct containing the up-and-down- fragments of the gene from Agrobacterium in a suicide plasmid (SacB, GmR) in E. coli S17. To obtain the deletional mutant I did bacterial conjugation between Agrobacterium and S17. The result of the first crossover seems fine since the Agrobacterium colonies I obtained contain the up and down fragments after I did the PCR check. However, in the second crossover, I thought at least I would see some colonies going back to wild type, but no bands are shown in all colonies I picked following the PCR. This experiment was actually already done by someone else in my lab, and she obtained the mutant. I used her mutant and the wild type of Agrobacterium as control. The PCR result of the control seems fine. So what is the possible reason for this failure, and is there any suggestion?
I'm now operating an experiment of MT genes amplification and sequencing. When amplifying the mt large subunit ribosonal RNA gene (16S), the agarose gel electrophoresis result showed me that there existed a smear bigger than the aimed band. Everytime when I was sequencing this gene I may encounter the same problem. So who can tell me what's the smear's component, and why it was mistakenly amplified?
(I use 16SAR/16SBR as the PCR Primers)
My goal for this experiment is to knockout a gene of interest in budding yeast (Saccharomyces Cerevisiae) and replace the whole gene with a G418 resistance cassette.
As a broad overview, PCR was used to amplify the G418 resistance cassette from a strain from a deletion collection which was then used to transform into a WT strain. After transformation, the transformants were plated onto YPAD for recovery then selected for using a G418 media plate.
After going through the steps provided in the protocol (attached below), colonies were able to grow on the 2xG418 plates suggesting potential candidates that were transformed to have the G418 resistance cassette. However, after molecular genotyping using PCR, it was found that the cassette was not located relative to the desired gene loci.
We expect the issue is related to the transformation efficiency and was planning on increasing the heat shock time from 7 minutes to 15 minutes and also improve the homology of the PCR product by using a mixture of Taq/Pfu rather than Taq alone.
Any suggestions with troubleshooting would be greatly appreciated!
Hello
I am doing multiplex PCR for SCCmec typing of MRSA, but I am not able to get a band which is 1791bp and it is the largest band while all other five bands are present as intended. I used DNA extracted through boiling for PCR and the primer and PCR conditions are well optimized used by many researchers. Any suggestion in this scenario please.
Hi everyone,
I'm trying to perform ATAC-seq without using any commercial kit, but it's been challenging to find a protocol that aligns with this approach. I’d like to ask if anyone has experience with this.
Here’s the workflow I followed:
1. Transposon Annealing:
ME A: 5'-(index sequence)AGATGTGTATAAGAGACAG-3'
ME B: 5'-(other index sequence)AGATGTGTATAAGAGACAG-3'
ME rev: 5'-pCTGTCTCTTATACACATCT-3'
By following the standard primer annealing protocol, I obtained two transposon oligos (ME A-rev, ME B-rev).
2. Nuclei Extraction: I followed the Kaestner Lab Omni ATAC protocol (file attached) and confirmed the extraction by performing MNase digestion. This step seems to be working fine.
3. Transposition:
I've attached the protocol for the Tn5 transposase I used. Following this protocol and the Omni ATAC protocol, I set up the transposition reaction with the following conditions:1 µl 10X Tn5 Transposase Buffer (final conc. 1X)
1 µl Annealed Transposon A (final conc. 1 µM)
1 µl Annealed Transposon B (final conc. 1 µM)
0.1 µl 10% Tween-20
0.1 µl 1% Digitonin
1 µl Tn5 transposase
Add D.W. to 10 µl
I added this reaction mix to the extracted nuclei and incubated at 37°C for 2 hours.
4. DNA Purification: I purified the DNA using the QIAquick PCR purification kit.
After these steps, I performed PCR for amplification and confirmed the product with gel electrophoresis, looking for bands at 300, 450, and 600 bp (corresponding to mono-, di-, and trinucleosomes, with ~150 bp added for index and adapter sequences).
This method worked once, but I haven't been able to replicate the results since then. Instead of the expected bands, I could only observe smears or odd bands after PCR (figures attached). I would really appreciate any advice or insights you might have.
Sincerely,
Hyelin
Dear Researcher,
I have used nested PCR to increase the specificity of Tetra Arms. Previously, I obtained results for another SNP using the same reaction conditions, and separate reactions (monoplex) verified that all primers are working efficiently. However, when I applied nested PCR, I observed no amplification; instead, the well glows. I have tried using a commercially available good quality ready mix and different PCR components, but the issue persists. I also tried changing the amplification temperature. Can anyone tell me the reason for this problem?
picture of gel is attached, well 1-6 = products from nested PCR, 7 = PCR product that was used for nestedPCR, well 8= ladder
Note: I am working on deletion polymorphism
Dear all,
I'm learning and taking the same genotyping PCR experiments and want to get / share some advice or experience on obtaining the right bands for the data.
I ran 3 samples of Stra8-iCre and 2 out 3 samples were not wild type, which was different genotype compared to the actual data from our laboratory and all of them were supposed to be wild type.
There was no contamination nor mistakes on performance of the protocol and I also regulated annealing temperature into 58 or 60 degrees Celcius for this whole times.
I wonder how can I get the right bands for Stra8-iCre and what was I missing except for the accuracy of the protocol and regulation of the temperature.
I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using original DNA plasmid as template for PCR. The PCR product contains 02 kinds of DNA which are in the same length (original template and newly synthesized product). PCR product will be treated by DpnI to digest all the original DNA. The remain PCR product (my target DNA, linearized structure) will be purified and performed in-fusion to circularized into plasmid, then transformed to E. coli for propagation. Plasmid will be extracted from the E. coli and confirmed by NGS. I repeat some experiments, unfortunately, it seems original DNA was still partly remain after DpnI or the site-directed mutagenesis reaction was not successful (I think) making new plasmid still identical to the original template.
Now I want to check whether my target amino acid is fixed or not before sending sample to NGS optimizing cost benefit.
I am having issues with the plasmid pDONR-P4P1R. I first conducted a BP reaction with this plasmid and saw a lot of background. To troubleshoot this, I performed a PCR reaction with M13F and M13R which revealed that some of the plasmids in my stock were missing the region between the att sites which contains the ccdB. I have tried replacing my lab's chloramphenicol, making new stocks, and making antibiotic plates at different concentrations of chloramphenicol but it is not resolving the issue. I have attached an image of my gel from the PCR for reference. Does anyone have any suggestions on how to resolve this? thank you!
I run PCR to checked presence of specific gene in shigella but I received 2 bands, one band showed product size expected and another one showed higher product size. I would like to check the my primer pair may have multiple binding sites or not. I did BLAST primer and got the result with 1 primer pair. How do I know that my primer pair has multiple binding sites through this result? Any other websites can check whether the exact position that my primers bind to the genome.
Your answers are really appreciated.
I am doing RAPD analysis to test for clonality in wild populations of three different plant species (Trientalis europaea, Cornus suecica and Rubus saxatilis). While I get quite good results for one of the species (Trientalis europaea), amplification fails for the other two.
I extracted DNA with the Qiagen DNeasy Plant Mini Kit and I tested 60 decamer operon primers of three different series (A, N and AF). I already optimised my PCR protocol in many ways (annealing temperature/number of cycles/PCR program, type of polymerase, amount of Mg, amount of DNA etc.), but without any success. Besides, since I had successful amplification for at least one of my species, the problem seems to be species-specific rather than protocol related.
Attached is a gel photo of my latest run which shows failed amplification for one of my focal species while my positive controls show clear bands, which suggests the problem is not due to PCR conditions. What am I doing wrong or overlooking?
I would very much appreciate any help.
I have done an extraction for DNA from amniotic fluid sample using Thermo Kit but when i measured DNA on the Nano drop I realized that it is contaminated sample as the ratio 260/280 is 0.7..I tried to make PCR with concentration 100ng in total volume 15 microlitre but no bands appeared. could you please tell me how can I increase the purity of sample and how to overcome this problem to get a successful PCR?
Thanks
Hello everyone,
I performed a PCR yesterday, and the results showed no bands on the gel. Of course, I probably missed some crucial steps, like adding my samples to the PCR strips themselves, for instance. However, I seem to remember not missing any steps in my protocol. One thing that I didn't do, however, was vortex my primer + master mix solution together after adding them to a 1.5 mL tube. I doubt this would cause me to have no bands, but would this really affect my results? Thank you!!
Hello all,
I have been trying to follow a 2-stage PCR protocol used to amplify barcodes of a large yeast library, as per Nyugen et al. (2022) - https://link.springer.com/protocol/10.1007/978-1-0716-2257-5_22#Sec7 (section 3.3)
I have been working on this for the past 1.5 months now and have tried exploring all the variables from bead ratios to buffers to primers to template concentration. Although the 2-stage PCR seems to work every time now, the resulting yield after 2 cleanup steps is terribly low and cannot be sent for bulk sequencing.
Me and my advisor - both suspect cleanup steps as there wasn't anything wrong with the PCR itself. There are 2 bead cleanup steps after every stage of PCR:
1st cleanup - So the primers don't carry over to the 2nd stage of PCR.
2nd cleanup - remove excess primers and other contaminants.
The image attached would give some context to what I mean by low yield.
I am preparing to run a lot of samples on a 384 well plate and I wanted to test if any well in my PCR machine is dead so as not to lose any data. I would really appreciate help with how I can do that. Thank you.
What does ‘preventing carryover contamination’ mean?
If they are used, is pre-treatment with UDG necessary?
Thanks :)
Why does BLAST results give 100% similarity to two bacterial species and only identified to the genus level?
I did BLAST of the two cellulolytic species and one lignolytic species of bacteria after 16S amplification using PCR. However, my results indicate that the lignolytic organism is 100% similar to two different bacterial species. BLAST have only identified up to the genus level in my cellulolytic bacterial species. Can anyone assist to explain what has happened?
I am trying to use overlap extension PCR to combine 2 linear PCR fragments around 1kb each. I amplified both fragments with overhanging primers with a 20 bp overlap between the two fragments. When I do overlap extension PCR, I just get amplification of the individual PCR fragments. I am doing a PCR reaction for 15 cycles without the primers, and then adding the primers that flank either end of the combined product for another 15 cycles.
Does anyone have suggestions for troubleshooting? The overlap region between the two fragments has a TM of 54, and the primers have TMs of 74 and 78. For the overlap PCR reaction I tried an annealing temperature of 50 and 55, and for the extension reaction I have tried annealing temperatures from 55-70.
Hello,
We found three packages of Illustra™ MicroSpin™ G-25 columns in the cabinet of an unused lab. They are very old but have never been opened. I have never used this kit before, and I couldn't fully understand what it is used for from my internet search. Is it just a simple DNA purification kit, or is it something more functional? I am interested in recombinant protein production. Can I purify my ligation product with this before cloning into bacteria, or can I purify my PCR product with this before ligation? In which scenarios is this kit indispensable?
Thank you.
We are in need of primers for a PCR protocol for chicken genome. We are looking at the COI barcode, 648-bp fragment of the 5' end of COI. Specifically Primers FalcFA, BirdR1, CO1_ExtF, CO1_ExtR.
Could anyone provide me with a source of where I can purchase these from?
I'm working on RT-qPCR optimization for gene expression. In melting curve analysis, the 5x primer negative control has a peak that resembles the samples peak but looks shorter. 10x and 2.5 have no curve, and when I applied the gel for verification, no product was seen. Why does 5x primer have this melting curve but no product in gel?
hi every one
I am making vector construction (for fusion proteins) and in this moment I wanna to amplification of ADAM17 prodomain with PCR. to yet, I couldn't amplified the ADAM17 prodomain with RNA extraction of Human fibroblast and PBMC (from Blood).
can you please help me how i can chosen the best source of this sequence for extraction of RNA?
thanks with best regards.
How to assign allele numbers and sequence type after amplifying housekeeping genes by PCR for genotyping of S. pneumoniae strains by MLST using bioedit ?
Dear Colleagues,
I have the following problem: I’m trying to amplify a cassette with a resistance gene (about 2 kb) from the yeast genome, so that I can then insert it into a plasmid. With Taq polymerase everything works. With proofread polymerase (Tersus) there is no PCR product. Everything works with the positive control (with ITS primers, the product is about 800 bp). I increased the elongation time to 4 minutes, increased the primary denaturation at 95 to 3 minutes. There is no the product. I would be very grateful for any advice.
I get two different bands from BCMV after PCR