Before I start cloning, I was just wondered whether somebody would be ready to share the plasmid?
Thanks in advance,
Regards,
Peter
We are repeatedly troubled with strange Protein bands in PAGE after PCR using Q5 polymerase from NEB. Who has measured the molecular weight of Q5 already and could help us out? Thank you all in...
31 December 2019 6,048 3 View
I am writing a script for Processing fluorescence spectra. In Order to make it useful for a large audience, I am collecting Sample Data of Export Files from fluorescence spectrometers of different...
03 April 2018 8,105 11 View
Dear all,I was able to identify a new, synthetic molecule which is able to induce expression in bacterial gene regulation (only a particular operon is effected) - The practical use of this...
11 December 2016 7,220 0 View
Dear all, after IMAC elution, my E. coli expressed protein (pI=6,32, MW=13 kDa) is in 400 mM Imidazol and 0,3 M KCl. TEV-Protease and cleaved off His-Tag are also swimming in this solution. I want...
05 June 2016 3,911 12 View
Dear all, my latest gel permeation purification produced some strange results (see chromatogram attached).I am purifying a small 14 kDa protein after Ni-NTA-affinity chromatography and subsequent...
04 May 2016 1,212 7 View
Dear all, my E. coli lysate is extremely viscous, to the point that it is impossible to apply it to the column for purification. What can I do?I added 500 units of DNAse to 50 ml, but the...
04 May 2016 9,746 8 View
Dear all, lately I get some serious contamination in my E. coli cultures. I suppose it is a fungus of some kind. I am using minimal medum with no trace elements and no vitamins, the only organic...
04 May 2016 9,078 46 View
Dear all,I am trying to purify recombinant prion protein (PrP).The chromatogram during gel permeation chromatography shows that nearly half of my protein exists in the fom of dimers. Running...
04 May 2016 10,064 4 View
Dear all, I am using ammonium sulfate precipitation of protein as a first purification step from my crude bacterial lysate.The method works well, the proteins precipitate in sequential order as...
04 May 2016 3,915 9 View
Dear all, we all use prepacked chromatography columns for affinity binding in protein purification (most prominent metal affinity like IMAC). I always wondered whether it would be better to load...
03 April 2016 644 5 View
I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...
08 August 2024 4,645 2 View
After immunohistochemistry of previously fixed in PFA and EtOH and then frozen 20 μm sections of zebrafish brain, DAPI staining is very weak (right) compared to the same sections stained without...
05 August 2024 9,637 2 View
Hi all, I need to introduce an ARS (autonomously replicating sequence) in my plasmid but I'm not sure which position would be the best. Does anyone have any suggestion? A picture of the plasmid...
05 August 2024 1,573 4 View
I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...
05 August 2024 9,059 3 View
I am performing ligation of the plasmid and a target gene. The steps I have taken are: 1. Double digestion of the plasmid and target gene 2. Ligation of the plasmid with the target gene 3....
05 August 2024 2,570 3 View
I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...
05 August 2024 9,798 3 View
I have tried several times to isolate lymphocytes from mouse spleen, but all attempts have been unsuccessful. I tried most available protocols. I used different dissociation media (HBSS with Ca...
04 August 2024 9,913 7 View
Hi, everyone. I want to transfer two genes in to the pseudomonas bacteria that isolated from the soil. The bacteria that I want to use for cloning haven't been identified and not be sequenced,...
02 August 2024 3,987 1 View
Dear All, I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on...
31 July 2024 9,892 4 View
Hello I am trying to create a stable cell line in HEK293 via Lipofectamine 3000 transfection. My plasmid is a CD63-IL10-GFP construct with Puromycin resistance. I am successful with the...
30 July 2024 6,648 1 View