PCR Machines

8 days ago

This post is about some perplexing and difficult to get rid of contaminant that accumulates in yeast, especially stationary phase samples, that inhibits PCRs in a hard to predict way. Now I'm no biochemist, so I don't have a solution for you, but I do have a dirty hack that should let you do large batches of extracts that a...

17 days ago

Hi all, This is the last post in a series of 4 in which I want to share a quick and easy to use procedure for engineering a yeast strain. The idea is to go from the cloning stage, right after doing a PCR (or order) to get the DNA you want to clone, up to the process of transforming the yeast and selecting the positive clon...

about 1 month ago

I am reaching out to inquire if anyone in the community has experience using dCasRx to target exons or introns, specifically in the context of observing changes in splicing patterns. If you have worked with dCasRx in this manner, I would be grateful if you could share your insights or any relevant resources. Thank you in ad...

2 months ago

I just stumbled upon an extremely cheap and simple way to clean up PCR products without the need for columns or fancy reagents. Here's my slight tweak of this wonderful Open Wetware entry. PCR Precipitation Buffer To a 15mL tube, add 3g PEG 3350. (I use Miralax brand which works well) Add 2.19g NaCl. Fill to 12mL with dist...

2 months ago

Hi all, I have been using this very very quick gDNA extraction (<5 min) method for plants for a few years now and wanna share it here in case you find it useful. I mainly use it for Mimulus, Aquilegia, but tried it in Arabidopsis too, most likely applies to many other plants: collect the tip (~ 1cm for small leaves, ~2cm ...

2 months ago

Hi all, I have made this post as the first of a series of 4 in which I want to share a quick and easy procedures for engineering a yeast strain. The idea is to go from the cloning stage, right after doing a PCR (or order) to get the DNA you want to clone, all the way to the process of transforming the yeast and selecting t...

almost 2 years ago

PCR can be performed directly from a scraped cell colony, using an extended denaturing step to cause lysis of the cells. This is a nice time saving protocol for PCRing targets directly from antibiotic selective plate media. Colony PCR with Patch Plating If subculturing via patch plate, divide a warm LB-Amp plate into a gri...

almost 2 years ago

The usefulness of PCR extends beyond replicating a target segment. Unfortunately, PCR cloning can be difficult due to the 3’ adenosine bases added to the ends of PCR products by Taq polymerase. This experiment details cloning a gene from S. cerevisiae such that it is compatible with the TOPO TA cloning process. These adenos...

Warnings, solutions, and mysteries regarding extraction/PCR from large amounts of yeast stationary-phase genomes

Warnings, solutions, and mysteries regarding extraction/PCR from large amounts of yeast stationary-phase genomes

Quick and easy bioengineering procedures: Yeast (S. cerevisiae) colony PCR

Quick and easy bioengineering procedures: Yeast (S. cerevisiae) colony PCR

dCasRx/13 and splicing

dCasRx/13 and splicing

Ultra-cheap PCR Precipitation using PEG and Salt

Ultra-cheap PCR Precipitation using PEG and Salt

Very quick and "dirty" (actually very clean) gDNA extraction

Very quick and "dirty" (actually very clean) gDNA extraction

Quick and easy bioengineering procedures: Golden gate cloning

Quick and easy bioengineering procedures: Golden gate cloning

Colony PCR

Colony PCR

TOPO TA Cloning

TOPO TA Cloning