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Questions related to Cloning
I am currently trying to work with the epithelial lung cancer cell line CALU3 cells for a transfection study and finding that my cultures are taking an extraordinarily long time to get to even 50%...
20 November 2020 4,364 7 View
Hi everybody, I have a problem with protein purification and I hope that I find the answer here. I used pET151/D-TOPO® for cloning and expression of three candidates. All of them have been...
18 November 2020 7,384 12 View
a) I have to perform the transformation, for which I selected the TOP 10 strain of E.coli, can i use other strains of E.oli as well? b) For making competent cells, We incubated the primary...
17 November 2020 8,477 3 View
My plasmid (pJET1.2) contains an insert of around 2969bp. The total size of plasmid 5942 bp. I am cutting it with AhdI, SgrAI and BssHII. Desired bands are of 2969, 1632 and 1432bp. I want to...
16 November 2020 4,868 4 View
I am planning to overexpress an sRNA in K.pneumoniae (inherently resistant to Amp, hence cannot use pUC19 because of it Amp resistance and not useful to scree). Which is better to clone in an...
15 November 2020 1,715 3 View
Dear all, How I can clone two genes in pcDNA vector, simultaneously (one of them is target gene with 500 bp length and one of them is GFP). Also, how we can transfect the vector into SH-SY5Y cell...
13 November 2020 6,236 3 View
I recently bought a LNCaP clone FGC (ATCC® CRL-1740™) vial and thawed it following all the steps described in datasheet. I used the recomended culture medium (RPMI-1640) and FBS at 10% (all from...
11 November 2020 5,390 3 View
Hi everyone .. I have great difficulty in cloning a promoter of a stem cell marker in an expression vector. the region is high GC percentage and in fact in the pcr, I had to use both adequate...
09 November 2020 8,806 3 View
Hi Everyone, I am trying to sub-clone TCF7 gene (transcript variant 1) into pCDH-CMV-MCS-EF1 Puro vector from some other commercially available vector by InFusion cloning(recombination). I got...
06 November 2020 6,429 2 View
Hi, I need to clone 30-40 PCR products. I tried direct sequencing of the products but found out that they are mixed and result in overlapping peaks if directly sequenced. I want to clone these...
06 November 2020 9,987 3 View
I am trying to amplify full-length of lncRNA (>2000 bp) that contains poly A tail for cloning into expression vector. However, due to the poly A tail in lncRNA sequence, the reverse primer that...
28 October 2020 7,817 3 View
HI all, I found toxin-antitoxin system of Mtb interesting through a invited talk at out Dept. I was wondering if the essentiality of the system has been investigated in Mtb. I would appreciate...
24 October 2020 3,823 2 View
Hello all, I am trying to introduce an annealed DNA fragment into the double digested vector. The ssDNAs were ordered without a phosphate group modification. The vector is digested with XhoI and...
21 October 2020 8,481 5 View
Looking for a way to import LogFc values from an RNAseq database to Cytoscape or String.app to create a string map. We would like to costumize the size and colours of the nodes for every pathway...
21 October 2020 6,320 1 View
I am going to clone a gene into pET32 a expression vector. After completing the cloning process, I verified my insertion by PCR an I see about 800 bp product . Then also, I wanted to verify with...
13 October 2020 6,997 2 View
Hey guys! I'm working on a huge complex, trying to purify it for structural studies. All of the components need to be cloned into one vector, and there are over 20 of different proteins in it....
13 October 2020 914 2 View
I am currently trying to clone a gene of 1500 bp into pet 28a by using the NdeI and XHOI restriction sites. These sites are introduced into my gene by overhang PCR. Restrictions are done for 2...
09 October 2020 5,705 7 View
Dear research community I am planning an RNA-seq experiment of iPSC-derived retinal organoids. The goal is to compare the effects of a gene knockout. I would like to know what would be considered...
06 October 2020 8,011 0 View
Hello, we have the possibility to send an eperiment to the ISS, I was thinking to clone various bacteria or cell cultures and to divide them in 6 samples, two will go for a month to ISS and be...
05 October 2020 2,530 3 View
Nowadays researchers are cheated by cloned journal sites. If anyone has such a list, it is good to share.
03 October 2020 3,539 6 View
I have tried cloning my gene into an empty vector and grow them up using E.coli competent cells and have no colonies. I also attempted to clone the gene fragment first using the TOPO TA kit and...
01 October 2020 4,617 7 View
Hello, I am trying to clone 3 genes into a commercial vector using restriction and ligation. I am attaching a small scheme of the cloning procedure. I amplify the fragments with PCR, then cut...
30 September 2020 8,878 8 View
Did anyone tried single-cell cloning from SkBr3 cell line. I am failing in growing cells post single cell seeding in wells. Any help is appreciated
28 September 2020 8,891 4 View
Dear All, I am trying to clone and purify codon-optimized viral proteins using pET28a expression system. Even though I have the perfect insert in the recombinant plasmid, there was absolutely no...
25 September 2020 3,770 8 View