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Questions related to Cloning
I am facing a problem with the plasmid from addgene (pcW57.1), when transformed into chemically competent DHFa cells, did not give any single colony overnight in ampicilin containing agar...
18 July 2020 8,136 1 View
I need to perform qRT PCRs on cDNA obtained from VERO E6 cell lines (derived from African Green monkey) and I need to choose ne (or more) housekeeping gene. I usually use B-actin and GAPDH for...
16 July 2020 7,055 0 View
Hi! Please! I need to preserve RNA at room temperature, the pellets. Is there any protocol for do it?
16 July 2020 2,320 1 View
i have inserted TP53 gene in mammalian expression system after cloning protein has been formed. now i want to detect exogenous protein that i have inserted so i want to know the approach to do it....
15 July 2020 333 6 View
I want to do antibody neutralisation assay with HIV pseudovirus and I am stuck in calculating 200 TCID 50 value of virus. Please suggest me.
12 July 2020 3,301 0 View
Standard practice for plasmid minipreps is to grow ~5 mL ecoli cultures overnight (~16 hrs). This works well and I always get good plasmids. However, a few times I wanted just *a little bit* of...
01 July 2020 733 8 View
I want to look at CD34 subsets in neuroblastoma pts, (bone marrow) before and after treatment. CD34/117, CD34/38, CD34/133, CD34/90 and CD34/41. I have clones for CD34, CD38 and CD117 but I cannot...
26 June 2020 4,194 2 View
Dear all, I am working on a whole genome sequence analysis of a Cyanobacteria strain. After sequencing I have got large number of Illumina reads (150 bp in length) and after trimming...
26 June 2020 5,982 0 View
Recently, I have been working with some genes contained in pBlueScript II SK (+) and that contain XhoI and XbaI restriction sites. I would like to clone them in PET28a. For instance, the...
23 June 2020 3,586 5 View
Hi, I've cloned my GOI successfully into pAdenoX-ZsGreen1 (TakaraBio). However, during the Restriction Enzyme digestion verification step, there is always an extra band on the agarose gel for...
19 June 2020 3,701 10 View
I am applying principal component analysis (PCA) on my data of morphometry but as the result of analysis I got just one eigenvalue greater than 1. So, is it possible to consider just one component...
18 June 2020 2,547 22 View
I used gateway cloning to clone my insert into the donor vector. After having colonies, I did a mini prep and ran it on the gel. On the gel I could see a clear band on the right length for my...
15 June 2020 9,913 3 View
I'm looking for an assay to test degranulation of mouse NK cells but all the protocols I find are for human NK cells cocultured with human K562 cells. Thanks!
15 June 2020 9,228 1 View
Dear All, I am working with thermo fisher H9 cell line, human induced pluripotent stem cell. I made a mutation on them with crispr cas-9 I obtain single colonies and I propagate my clones. I...
14 June 2020 9,136 3 View
I need to amplify a pcDNA3 plasmid containing human SCN9A CDS insert (pcDNA3-SCN9A construct). The CDS is around 6 kb, and the total construct length is approximately 11.2 kb, which is a bit...
08 June 2020 2,253 2 View
im comparing a sample based on the analysis of its ITS1/2 regions with the BLASTn tool for my internship. What's the maximum homology percentage required to determine if my sample at hand is a new...
04 June 2020 5,984 5 View
Hello, I am working on isolation of polar coumpond. I make my reaction in water using tris buffer 0.5 M , my coumpound that i want to isolate is water soluble. I was thinking to use gel...
28 May 2020 8,398 0 View
Hi all. I'm trying to create a heterozygous deletion (55bp) mutation in iPSCs to model malignant tranformation. I'm using two sgRNAs in a plasmid based system (puromycin selection). I obtained ~20...
19 May 2020 5,458 1 View
As of now, there are over a million people who have recovered from COVID all across the world. If some of their antibodies were to be isolated, and antigen binding specificity established, I...
14 May 2020 5,413 4 View
Hi, Does anyone know of any plasmid where I can clone two genes under two promoters? Basically, I want to coexpress these protein together but with Arabinose inducible system. pETDuet,...
08 May 2020 8,277 0 View
Hi Everyone, We are having some difficulty doing E. coli cloning because our insert is toxic. We thought of using Rosetta E. coli (expression line) for cloning since it exherts tight control...
30 April 2020 6,648 7 View
I trying clone for almost 2weeks through Gibson assembly? quick my procedure: vector : 5103bp conc = 22.2ng/ul insert: 387bp conc = 58.6ng/ul I was trying to ligate with total of 10ul. I...
30 April 2020 1,926 0 View
Hello, I have to clone 3 pieces together: 1. 2 Pcr made- the inserts. One 4200bp (13 ng/microliter), one 460bp(21ng/microliter) 2. Vector 3800bp(13ngng/mictoliter). All pieces were cut with...
28 April 2020 900 3 View
Hello everyone, I have problem about one blunt end and one sticky end ligation. I'm trying to ligate insert size 2,017 bp and vector size 5,300 bp. The PCR product (Insertion, ~2,017 bp) has...
27 April 2020 5,384 0 View