PCR Cloning

2 months ago

I've been having trouble amplifying a 3kb promoter region that's AT-rich (80%!) w. lots of repetitive elements, then I saw this paper: https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-017-2982-1. Gave it a go without much hope, but lowering the extension temp and adding some MgCl2 instantly did the miracle! I...

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...

A potential hack for amplifying AT-rich & repetitive regions

A potential hack for amplifying AT-rich & repetitive regions

Colony PCR

Colony PCR

TOPO TA Cloning

TOPO TA Cloning