Why is my protein precipitating after isolation?

My protein is 28kda and is some what hydrophobic with a pI of 6 and has a his-tag. We typically purify in 1x TBS with low salt at pH 7.5. During dialysis and or concentration the protein...

18 September 2020 6,493 11 View

What is the best way to transition media for hiPS cell culture?

Hello I have human iPS cells that are being thawed and cultured in mTeSR1 media. I want to transition them to E8 media. In the past we have had very limited success with this type of media...

31 October 2017 1,814 4 View

What is the correct method/formula to calculate the p-value for qPCR data?

Please explain how the p-value and SD is calculated using Cp values.

15 July 2014 7,870 3 View

Difficulty in cloning of 1kb gene into vector?

I am facing difficulties in cloning a 1kb gene into a vector (pJIT163). I have my gene on interest (GOI) in pUC57 and want to clone in pJIT163 using SalI and BamHI restriction sites. I am getting...

25 February 2021 3,221 7 View

Is it harmful to breed for Cre Homozygosity initially?

Hello, I am currently using a Cas9 knock in system that will be expressed in the liver. To do so STOP codon fl/fl Cas9 KI mice were bred with Albumin-Cre mice to generate heterozgous pups, that...

24 February 2021 5,759 3 View

Alternatives to traditional cloning of insert into backbone (dsDNA)?

Hi, I am stuck with a cloning experiment: The insert & backbone(bb) share one compatible end at 3' end(NotI) and one incompatible end at 5' end (XbaI for insert; HindIII for backbone). So i...

16 February 2021 1,664 3 View

Can part of recombinant protein not be expressed?

Hello. I am currently trying to express a recombinant gene with Avitag at the N-terminal. I'm aiming at a final product that looks like this: Avitag-(target protein) The protein I use is already...

14 February 2021 2,039 4 View

Transformation into the Stbl3 chemo competent cells?

03 February 2021 1,111 5 View

How best to model transportation during usage phase of LCA for a mobile water tank in SimaPro (kg*km)?

I would like to model the usage phase of a mobile water tank. The tank is transported on a tractor or lorry so I need to model the transportation too. My problem is that when I select a...

01 February 2021 6,759 3 View

What questions should i include in my questionnaire?

I am writing my dissertation, about adults who trained at elite status in their youth, i am constructing a questionnaire with the goal of seeing, how many sustained injury, how many sustained...

28 January 2021 4,787 3 View

What is the best way and technique for serial dilution of bactria to get accurate number of colony ?

Hello I am infecting cells with bacteria in a well and then I am doing serial dilution of this well 1 in 10 and culture the two last dilution on MHA agar I have difficulty in getting accurate...

26 January 2021 9,256 5 View

Good day, I have not been able to amplify the plasmid Frt-hspB1. Somebody could give an advice me?

I acquired the plasmid in Addgene, reconstituted the filter paper where the plasmid comes from with 30 microliters of TE buffer pH 8, immediately afterwards perform the transformation of E.coli...

25 January 2021 2,526 3 View

Has someone used TTC agar overlay method and can help me?

I have tried to use TTC agar overlay method for a few times to identify respiratory-deficient yeast which were affected by EthBr. The problem is that cells which should have stayed white also have...

22 January 2021 585 10 View