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

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

Why and how to avoid negative values in optical density by spectrophotometry when measurements corrected by blank solvent?

I am trying to assess chlorophyll-a content from coral tissue using spectrophotometry (with SPECTROstar Nano, BMG). However, I always seem to obtain some negative values in my optical density raw...

20 January 2021 1,542 4 View

I am having trouble with cloning my gene. How can I fix this?

16 January 2021 6,566 8 View