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Questions related to Cloning
Hello, Does anyone know about cheap services for library preparation of small RNAs from biofluids? It may also include RNA extraction/sequencing and can be outside North America/Europe. Thanks
31 August 2021 6,432 3 View
Hi, My gene of interest amplifies at a Ct range of 22-26 cycles. I found that my no-template control amplified at a Ct= 28 while the other NoT duplicate amplified at a Ct=34. Should I be...
25 August 2021 610 1 View
I am using Qiagen RNeasy Midi kits to extracts RNA from various rat brain ROIs. I have a lot of samples to process, and would ideally like to do the tissue homogenisation step separately from the...
10 August 2021 1,097 0 View
Hi everyone, I am hoping to overproduce an antibiotic by swapping promoters. I am just wondering what the advantages/ disadvantages using inducible/constitutive promoters for the overexpression...
03 August 2021 4,251 2 View
Hello everyone. I was trying to clone a region of ULBP1 and MICA gene from human PBMC cDNA preparation. I had setup first PCR and even though I got the amplification, both of them are at wrong...
23 July 2021 3,295 6 View
I have been using Prolong Gold with DAPI for my IF staining. However the last few times no DAPI signal has been showing up. Only problems I can think of is that I let the gel curate at 4C and...
22 July 2021 8,328 1 View
A probably silly question to phage experts. Is it possible to determine whether a phage genome is linear, circular, or circularly permutated using Illumina sequencing data and genome assembly?...
22 July 2021 1,718 2 View
Hi RG community, I'm about to order some single guide RNAs (gRNAs) for CRISPR/Cas9 site-directed knockouts following Bassett and Liu (2014) protocol (paper below). Briefly, two partially...
08 July 2021 8,509 6 View
how to decide which his tag to use between N & C terminal. and do orientation of gene of interest get affected by his tag?
07 July 2021 5,038 3 View
I am studying the metabolites of a drug in the cells and their supernatants and for this, I seeded my cells in a 96 well plate with the treatment. Now I want to transfer the cells in Eppendorf to...
02 July 2021 6,759 3 View
Are there any disadvantages pETDuet-1 vector to do coexpression, especially for purification step.
22 June 2021 6,781 0 View
I normally use a nested PCR on 16S-23SrRNA region, for diagnosis of a plant pathogenic bacterium. It is a well-established and almost “trivial” protocol, shared in many laboratories. Recently when...
07 June 2021 2,987 10 View
Dear community, I did a tetrad dissection of yeast where I (hopefully) mated two markers into one stain. To be sure I selected real tetrads and to determine their mating type I did another mating...
07 June 2021 1,354 0 View
Hi all, I intend to create a point mutation in the genome of C2C12 cells but am having difficulty deciding what ratio's of gRNA plasmids, Cas9n plasmid, and ssODN repair template. I am...
30 May 2021 5,119 0 View
I want to generate periplasmic protein of another bacteria in E. coli BL21 strain ? How can i do this ? I verified the cloning with pcr but protein production is not happening. Anyone have a...
30 May 2021 4,593 3 View
I have just completed an antibody library based on phage display. However, it's only about 10^10. It was my first time to construct a phage library. It seems that I missed something during the...
28 May 2021 260 6 View
hi all! Could I transfer my cell culture I've already seeding in 12 well plate and treaded with some reagents in CO2 incubator, into 96 well plate to read the results by plat reader which couldn't...
24 May 2021 7,427 2 View
I have proteomics data that have missing values. What is the best way to impute the data if my data is not normally distributed? I have 4 treatment groups, and I have scenarios where one...
24 May 2021 231 1 View
Dear Researchers While I am reconstructing the phylogenetic tree, I am getting an error message like this "Aligned sequences must be equal lengths (in line 3133). There was no error in the...
18 May 2021 2,220 5 View
Hello, everyone, I am using rats lung tissue to do the hydroxyproline assay, after adding the DMAB reagent and set the plate under 65 degree C for 30 mins. The color of the experimental wells...
14 May 2021 531 0 View
Hi I am growing HepG2 cells expressing NTCP receptor at a high level under 500ug/ml of G418 At the same time I want an efficient knockdown of one gene and ready to do Puromycin selection. I am...
14 May 2021 476 2 View
Is it possible to transduce a human CAR into mouse T cells and have the mouse T cells function when the CAR binds it's antigen? I am thinking that if anything the singling domain of the CAR, as it...
11 May 2021 6,128 2 View
I need to isolate fibroblasts from a neonatal, in order to culture them. I have some conciderations about the method of isolation (digestion or explantation). Thank you very much in advance.
06 May 2021 3,114 2 View
I am using the NEB Monarch Genomic DNA Purification Kit to extract gDNA from BL21(DE3) cells. The protocol says to use a maximum of 2x10^9 cells but does not mention a recommended culture volume...
26 April 2021 4,144 2 View