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Questions related to Cloning
Hello to all researchers I would like that someone can guide me if I can validate my results. I tested the activity of a positive clone which produces a lipase (metagenomic DNA) directly in the...
27 April 2020 3,553 0 View
Hello everyone, I am writing a report looking at human sexual selection and how there is a difference in the preferred age of potential mate between sexes. I collected 104 lonely hearts...
17 April 2020 1,159 3 View
I'm trying to install GATK for my SNP analysis but when I tried to install GATK it appeared an error consisting: A problem occurred evaluating root project 'gatk' Could not get unknown property...
15 April 2020 3,648 3 View
I would like to clone a big region (50kb) of DNA.
09 April 2020 5,889 5 View
I have been using the NEB Hifi Assembly master mix and following the same protocol. I used three different constructs but the same vector. Whenever I do the colony PCR for screening, I always get...
31 March 2020 7,949 3 View
I'm trying to clear up a question that I have answered and no useful answers showed up here.
31 March 2020 8,376 3 View
I use phagemid pCDisplay3 and M13KO7 to construct Human Naive phage library. before digested with SfiI , I prepared ScFv (750bp) using gel purification. After electronic shock , ,plating on...
27 March 2020 4,106 2 View
I inserted 4 different genes in vector, and cloned in E.coli. when I grow them on IPTG containing plate, Each bacteria grow different. If I pick each bacteria with different number, then each...
27 March 2020 4,613 3 View
Hello! I would like to clone a very large fragment of DNA into my vector (which will be digested with restriction enzymes) . I will need to amplify this fragment by PCR in pieces (2 or 3) and then...
27 March 2020 4,948 2 View
Hello, I have inserted a 580 bp sequence in 3xFLAG plasmid. This sequence has a 66% GC content. PCR annealing temperature was high at 68C. Purification, digestion were fine. Transformation was...
27 March 2020 411 3 View
We tried to knockout a gene of our interest by CRISPR/Cas9 using lentviral approach. We transduced cells with lentivirus containing Cas9-gRNA-puromycin, selected with puromycin for 5 days, then...
26 March 2020 4,889 2 View
I did cloning. it was like a traditional approach. Firstly, Restriction digestion was performed for both vector (8kb) and target gen (700bp), and also the vectors and target gene were run on the...
22 March 2020 9,724 4 View
I have encounter countless problems along the methylation analysis way. Most current is with sequencing of plasmids after GW cloning of PCR product. The PCR product comes from bisulfite converted...
22 March 2020 1,169 6 View
Previously, I had read a mechanism about conditionally-skip transcription from a cloned cassette in an expression vector. But i can't find that paper. It was containing a cis-regulatory element as...
21 March 2020 3,359 3 View
Vectors and inserts were ligated after cutting with EcoR1 and Xho1, respectively. The colonies were picked and cut again with restriction enzymes. But after cloning, I saw the bands of the...
21 March 2020 636 4 View
I'm going to be sub cloning CHO cells but I need some advice on what to look for. These clones will then enter an AMBR 250 for selection of best performing clone, where we will pick the best and...
16 March 2020 2,248 2 View
Hi, I have cloned my protein with TEV protease site (His tag-TEV site-linker-protein). I purify my protein with Ni-NTA and then do on column TEV cleavage (overnight). I hardly see any TEV...
11 March 2020 1,431 3 View
Hello, all. I've been unsuccessful in cloning a 6.3 kb gene into pGEM T easy. Are there other plasmids that are optimal for cloning an insert of this size? Alternatively, is there a protocol...
10 March 2020 1,470 1 View
I keep reading that cDNA obtained with random hexamers yield short cDNA fragments, which are fine for qPCR, while for longer targets, oligoDT should be used. However, shouldn't these short...
09 March 2020 6,663 4 View
There is a recent publication titled "In vivo DNA assembly using common laboratory bacteria: A re-emerging tool to simplify molecular cloning" (J. Biol. Chem, 2019; doi:...
06 March 2020 263 3 View
I would like to conduct a "rescue" experiment in a knock-out cell line that involves designing/cloning a plasmid that expresses my gene of interest and also confers Blasticidin (or G418)...
06 March 2020 1,639 0 View
I am trying to create a library of promoters to test them on my enzymes. I bought two kits from addgene which contained the promoters. However, for the same Promoter, i realized one has the kozak...
02 March 2020 4,354 3 View
Does anyone having experience with using Phusion Hi-fidelity and KOD hot-start Hi-fidelity DNA polymerases and what is the difference between these two. while i have used phusion for amplifying my...
27 February 2020 2,657 3 View
Respected Sir Hope you will be fine and in good health. I am working on role of Secondary metabolites collected by GC-MS from Syringa Oblata shrub with flea beetle interaction/defence Mechanism....
27 February 2020 5,206 4 View