Gary Robertson

9 Questions 8 Answers 0 Followers

Questions related from Gary Robertson

How did my insert vanish from my plasmid?

Short version: My plasmid contained insert but later after a second transformation into a second cell line the insert vanished and does not show up on PCR or sequencing Long version: I used...

22 February 2018 7,836 8 View

Can I use the short Kozak Sequence ACC before the start codon ATG for mammalian expresion?

The kozak sequence is GCCRCCATGG With the +4 G and the -3 R (either an A or a G) being the most important nucleotides. I can't control the first nucleotide of my protein after the start codon...

13 December 2017 3,539 0 View

How can I delete a large DNA fragment from my gene of interest using PCR techniques?

I have a gene of interest approximately 2000 bp long coding for a protein approx. 670 amino acids in length. I wish to excise one of the proteins domains from the gene. To do so I need to remove...

20 September 2017 2,779 5 View

What is the detection limit for a species (in time) of absorbance spectroscopy?

How long must a species last for, for it be detectable through typical absorbance spectroscopy. Say I have an enzyme with a turnover number of 300. Each reaction would take only 3.3 milli seconds....

11 August 2016 4,738 3 View

Do cumulitive effects on specific activity indicate a concerted mechanism?

I am looking at an enzymes reaction mechanism. I have investigated 2 mutations. both mutations individually reduce the enzymes catalytic activity. The 2 mutations combined render any product...

28 April 2016 6,754 9 View

Using ITC to calculate the Kd of ligand binding, when can one fix the stoichiometry of binding?

I have performed ITC to determine the binding affinity between a protein and its ligand. The ligand binding is weak and I don't get a full Sigmoidal Curve, instead it looks like a Hyperbola. Based...

27 January 2016 3,904 6 View

Can you relate the energy of enzymatic steps to Kcat?

If I know that the free energy barrier of the first step of a enzymatic reaction has increased (And I know by how much) but the other steps remain unchanged may I infer from this any kind of...

23 January 2016 8,693 6 View

Can anyone help with multiple bands after DNA double digest?

I am doing an Nde1 and BamH1 double digest. The gel is as followed Pet-28a with no insert (undigested), pet-28a (digested),Thermoscientific zip ruler express 2, pet-11a with insert (undigested),...

17 July 2014 2,162 6 View

Can anyone help with problems with non linear elution pH gradients during Chromatofocusing?

I am using GE healthcare poly-buffer 96 and PBE 94. I use a 0.025 M ethanolamine start buffer pH 9.4 and an elution buffer of PB 96 pH 6.0. I take care to degas all my buffers, keep the...

17 July 2014 4,115 2 View