Gary S. Laco

11 Questions 327 Answers 0 Followers

Questions related from Gary S. Laco

Terrific Broth and protein expression in E. coli, induction OD?

when using E. coli BL21 pLys, grown in Terrific Broth, for toxic/non-toxic protein expression I normally induce protein expression at ~0.5 OD using IPTG, and the resulting induced cultures double...

02 February 2016 799 14 View

How do you run protein samples in 6M GuHCL in SDS/PAGE? Here is a no heat method.

Here is my updated protocol that does not require heating the protein sample in 6M GuHCl (3/2016): Take protein sample in 6M GuHCl and shear DNA with a needle/syringe, then take 10 uL and add to...

05 May 2014 4,607 10 View

Can anybody explain why in His-tag purification resins most of the protein is not coming off resin with 500 mM imidazole?

I have used two different His-tag resins in batch mode to purify at His-tagged protein. The protein is bound to the resin in 6M GuHCL pH 7.9, and eluted with 500 mM imidazole pH 7.4, with both...

05 May 2014 4,297 8 View

What kind of competent cells do you use for cloning?

I was doing a standard sticky-end cloning using electrocompetent cells and for some reason all the cloned inserts were larger that the gel purified DNA fragment (used methylene blue to stain the...

05 May 2014 5,250 5 View

Megaprimer PCR that mutates 75 bases or more?

I have used the megaprimer PCR approach in the past to make mutations with no problems, however, the mutagenic primers typically mutated only one or two codons and now I want to mutate at least 25...

03 March 2014 9,951 5 View

Has anyone ever cloned short DNA fragments under 100 bp into a plasmid?

I want to clone a short DNA fragment of ~80 bp. The DNA fragment will be made by annealing two oligos together that were designed to generate 5' and 3' overhangs for the RE site, so the DNA...

02 February 2014 6,253 7 View

Is there a software program to add restriction sites to protein coding sequences without changing protein coding sequence?

I am looking for a software program that can analyze a protein coding region of DNA and find where restriction sites can be added without changing the coding sequence. I did find the program...

02 February 2014 6,107 17 View

How to quantify a native 8-mer peptide in solution over time?

I am planning an experiment in which there will be a large protein (~65 kDa) and a small 8-mer peptide in an aqueous solution. I want to monitor and quantitate the two over time. I will use...

12 December 2013 2,975 10 View

What is the correlation between in silico docking experiments; in vitro activity (cell-free enzyme assay); and cell culture based activity?

In some ligand/enzyme systems I have found very good correlation between the in silico docking of ligands in the active-site of an enzyme (i.e., calculated interaction energy score) and the...

10 October 2013 3,223 6 View

What do you use to stain SDS/PAGE protein gels?

Have been looking for a fast sensitive way to stain SDS/PAGE gels, but they all required 3x washes in DI water (other than the old Methanol/acetic acid Coomassie method). Have used the stain...

06 June 2013 3,107 93 View

Do you need to dialyze cytoplasmic lysate to remove free biotin when purifying in vivo biotin labelled proteins from insect cells?

I am looking at biotinylating a protein in vivo in insect cells where the insect cell media will be supplemented with 4 uM biotin. The protein accumulates in the cytoplasm. The question is: has...

01 January 2013 3,469 1 View