Daniel Fenando Paulo

5 Questions 19 Answers 0 Followers

Questions related from Daniel Fenando Paulo

Is PCR with M13 primers a valid way to test for complete double digestion of a plasmid?

Hi all, I made a double digestion of a plasmid (1 ug) using 20U of each EagI-HF and NcoI-HF REs and 1x rCutSmart buffer in a 50 ul final volume. The reaction was incubated at 37 ˚C overnight. On...

26 October 2021 8,991 2 View

Gel verification of Gibson assembly reveals low efficiency, but why?

Hi everyone! I performed my very first Gibson assembly (1 vector and 2 fragments) using the NEB Gibson Assembly Cloning Kit (#E5510S) and the assembly efficiency was quite disappointing as...

25 October 2021 6,886 11 View

What is the optimal Tm temperature for overlaps in Gibson Assembly?

Hi RG community, I'm designing primers for Gibson assembly with ~40 bp overlaps between adjacent fragments. However, I notice in SnapGene that the expected Tm's for those overlaps are quite high...

18 August 2021 4,658 12 View

Are PAGE purified oligos necessary for CRISPR/Cas9 mutagenesis?

Hi RG community, I'm about to order some single guide RNAs (gRNAs) for CRISPR/Cas9 site-directed knockouts following Bassett and Liu (2014) protocol (paper below). Briefly, two partially...

08 July 2021 8,525 6 View

Free softwares for genotyping in UNIX system

Hi guys, Does anyone know good FREE software for peak-calling (genotyping) of STR microsatellite markers (such as GeneMarker, OSIRIS, Geneious, etc ...) that runs in UNIX/Linux based system? I...

01 January 1970 2,502 4 View