Brendan Delange

5 Questions 14 Answers 0 Followers

Questions related from Brendan Delange

How can I get P1 Transduction working?

I've had difficulty producing a lysate from a strain of E. coli in which I have engineered a marked allele, for transfer to another strain.First, some background on the experiment:I have used...

02 February 2015 2,826 11 View

How can I increase the specificity of long-tail PCR primers?

I'm using tailed primers: 20-nt of priming sequence followed by a 10-nt tail. I know to use the annealing temperature (T_A) of the priming sequence (not the full 30-nt sequence) when designing my...

12 December 2014 914 8 View

How long can un-purified PCR reactions be stored at -20C after thermocycling?

The polymerase system I am using is Thermo Phusion Hot-Start II, a pfu-based recombinant Pol with a reversible affibody. The reason that I don't want to purify the reaction mixtures after...

12 December 2014 7,841 4 View

How long is the initial denaturation step for colony PCR?

I am performing colony PCR using Thermo Scientific's Phusion Hot-Start II polymerase. The manufacturer's instructions recommend an initial denaturation step of 98C for 30s. Should I increase the...

12 December 2014 6,527 10 View

How can I optimize colony PCR yield from single-copy genomic targets?

I am amplifying a 1.233-kb region from the genome of an E. coli K-12 W3110 subtype. For the reaction, I prick a single colony with the tip of a micropipettor and suspend it in 50 uL of nuclease...

12 December 2014 7,611 14 View