Polymerase Chain Reaction

In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short... | Contact experts in Polymerase Chain Reaction to get answers

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Questions related to Polymerase Chain Reaction

How long can agarose gel slice containing PCR product be stored?

I have agarose gel slices with PCR products in them for purification and sequencing. I need to send the slices to another lab for processing which may take up to 5 working days. Is it okay to...

04 July 2022 745 4 View

What is optimum temperature for the final extension in PCR?

As per literature generally, a temperature of 72°C is used for the final extension of PCR but does it has an effect on the final amplication results?

29 June 2022 6,966 3 View

Why is the product of my nested PCR bigger than the size of the first amplicon?

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...

28 June 2022 8,902 3 View

Two sgRNAs 500bp apart --> deletion?

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...

25 June 2022 9,523 3 View

Inconsistent PCR profile?

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...

24 June 2022 5,671 3 View

Problem with the pH in PCR purification using Qiagen kit ?

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...

21 June 2022 3,612 2 View

Why did the protein containing MBP-tag aggregated and precipitated when cleaved by Tev protease?

Hi，everyone， I am purifying a protein containing the contrust: N terminal-10×His+MBP+Tev site+protein-C terminal. The binding buffer is 50mM Tris-HCl, pH7.5, 500mM NaCl,10mM imidazole, 5%glycerin,...

20 June 2022 6,926 3 View

How to convert transcriptome data to protein??

I have transcriptome data and I am trying to do pathway analysis. I need to convert my transcripts into protein, can anyone suggest any software? Or any suggestions related to pathway analysis...

19 June 2022 1,722 4 View

Why am I getting non-consistent results from my assay?

Hi. I am recently trying to measure NAD+ from E2ND-100 kit from bioassay but I can't get any consistent results. This is my protocol. 1. Prepare 2 x 105 of A549 cell pellet washed with DPBS 2...

15 June 2022 166 0 View

Why aren't the bands typical in this agarose gel electrophoresis?

https://imgurl.ir/uploads/o756979__-1.jpg 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.

14 June 2022 4,734 6 View

I have used Hot start Master Mix for PCR. But I get multiple bands after running the gel. What could be the reason for this?

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.

14 June 2022 9,265 8 View

What is the reason for these kind of bands of my PCR product?

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...

14 June 2022 2,529 3 View

Optimal DNA template concentration for PCR?

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...

13 June 2022 8,728 2 View

Calculating the frequency of every pathogenic germline variant in every disease cohort?

I am trying to calculate the frequency of every pathogenic germline variant in every disease cohort. For ex: for variant 1:17588689, there are 20 het.variants (column H), I need to report what...

13 June 2022 6,940 0 View

PCR products from Yeast DNA (ITS) are not intense?

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...

12 June 2022 476 2 View

Why is my absorbance value of 260/230 close to 0 after purifying the PCR sample?

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...

11 June 2022 9,865 4 View

Site directed mutagenesis: Why there is No colonies in transformation?

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...

09 June 2022 8,887 7 View

Cos-7 express APOE ?

Hello everyone! My lab and I are wondering if COS-7 cells express APOE. I found this website :...

08 June 2022 5,875 0 View

How do I determine the genetic sequence of a mouse model?

I have recently obtained a mouse model B6;129-OoepGt(139A2-3)Cmhd/Mmucd from MMRRC https://www.mmrrc.org/catalog/sds.php?mmrrc_id=31076 I am having trouble getting our genotyping protocol to work...

07 June 2022 3,874 1 View

What is the different method of cleaning DNA?

After CTAB extraction I got 210 ng of DNA concentration and the ratio values were less than 1.7 (260/280) and less than 1.8 at 260/230 ration.

07 June 2022 8,801 0 View

Problem faced DNA quantification from leftover of TB positive samples for illumina sequencing?

Hi I am working on a project where I have specific segments of interest. Almost 18 region of TB positive samples were selected from where the mutation can be occurred. For that mutation the...

07 June 2022 693 3 View

How can I find the best set up for real time PCR with Taq polymerase? and what is the best anealing temperature?

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

06 June 2022 9,159 2 View

Can we dilute 2x PCR master mix or use it directly without any dilution ?

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

05 June 2022 1,757 5 View

What are tools for Genome Assembly of WGS FASTA Sequences?

Hi, I have 300 FASTA sequences of WGS of ameba. I need to assemble the whole sequence. Are there any tools (webservers or any command-line servers) available to combine the whole genome? Also Is...

29 May 2022 4,426 3 View

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