Hello all, I am performing PCR on about 1000 bacterial isolates (lysis solution only, no DNA extraction) for a culturomics project. PCR will amplify V1-V3 of 16S gene and then sending these out for sequencing to identify each isolate. I am searching for alternatives to running a small aliquot of each PCR product on a large...
Henrik Ellinghaus
4 months ago
PCR (Polymerase Chain Reaction)
PCR is likely one of the most useful techniques in biology labs. It was invented by Kary Mullis in 1984. Nowadays it helps us especially if DNA material needs to be amplified to work with higher quantities, but the applications of this method are almost endless.
Each PCR protocol uses the same standard ingredients. The pro...
Intro
PCR is definitely one of those things in science that borders on magic and witchcraft. I like to think I know what I am doing, but I still have those days even in my postdoc that I have to pray to the PCR Gods to get something to work.
In my experience, PCR success is mainly dependent on the primer set that you use....
Benchmarking CUT&Tag against ChIP-seq: https://www.biorxiv.org/content/10.1101/2022.03.30.486382v1
https://www.nature.com/articles/s41467-019-09982-5
Lovely blog post on CUT&Tag: https://www.activemotif.com/blog-cut-tag
The improved sensitivity of CUT&Tag compared to ChIP-seq is due to the use of pA-Tn5 to streamline lib...
I have set up a very small lab for identification of human respiratory viruses for educational and research purposes. After some trial-and-error, the following is what I've found to be the simplest and most cost effective. It should go without saying that biosafety protocols and appropriate regulations must be followed. Fee...
We recently collected genomic DNA from a screen. We did PCR using about 3-5ug of DNA per well on a 96-well plate and got 5ml of PCR product to purify. So what to do next to purify the 300bp PCR band for Hi-seq?
Do you do PCR purification followed by gel separation? I was wondering whether the large amount of genomic DNA ...
Indigo Minotaur
about 1 year ago
PCR before NGS Library Prep
I'm working on preparing a small CRISPR KO screen looking at a subset of genes. I'm following the Joung 2017 protocol using the lenticrisprv2 backbone and I'm having issues with the NGS step 58.
Since my screen is fairly small I'm only harvesting gDNA from 250,000 cells which should provide sufficient coverage. However, I'...
I would like a clarification regarding your Nat Prot 2017.
I have 10 different samples of gDNA that I will amplify for NGS library preparation and I will use different reverse primer for each one of them.
As you recommended in previous threads in this group, following PCR, I should pool all the PCR reactions for each expe...
Brown Kappa
about 1 year ago
Low DNA yield after gel extraction
I want to know if it is common to get low DNA yield after gel extraction? I get a yield of 5.8 ng/ul of extracted gel product from a 50ul PCR product loaded with 2ug DNA (using QIAquick Gel extraction kit suggested by 2017 protocol).
I am using LentiGuide plasmid and can see a correct band size at around 350 bp, though not...
Chartreuse Faun
over 1 year ago
CRISPR gene deletion
I want to delete a gene using a Transient CRISPR method. I have few queries regarding gene deletion:
1\. I want to use single gRNA with high off and on target score. How efficient it could be or I can use 2 gRNAs for better efficiency.
2\. I want to use 3 stop codons followed by restriction site and marker gene. Is there ...
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