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Questions related to Molecular Biological Techniques
I prepared a mastermix for technical triplicates for SYBR green assay (reaction volume 10ul) Out of the triplicates, usually the 3rd replicate will have difference in Ct value i.e. later Ct (range...
11 February 2017 3,270 3 View
I want to clone a 7 kbp pDNA with a 1.2 kbp fragment and I have already tested the 1: 3, 1: 1 and 3: 1 ratios but I always get self-ligation of the vector. How can I resolve this or improve the...
11 February 2017 2,327 4 View
I ran samples (looking for c.bovis in swabs and tumors) using the same primes/probes, Taqman Master Mix, DEPC H2O that was used last week on samples that worked fine. These new samples that should...
10 February 2017 2,868 4 View
I want to construct a vector to drive shRNA expression by U6 promoter. However, due to limited genetic space I would like to use minimum possible nucleotides of U6 promoter. Do anybody know...
10 February 2017 4,995 2 View
I'm having a hard time finding a reference for my specific conditions, any help would be appreciated: I have dsDNA bound to streptavidin-coated magnetic beads via a biotin interaction. The dsDNA...
06 February 2017 1,629 1 View
Hi everyone, I need a solution for an issue that I have with my AFLP protocol.Currently, I've been trying to make some AFLP's from a grass species that I study for my research project. However,...
30 January 2017 9,719 3 View
i did cloning for cytochrome P450- 2D6 into pGEX-6P-2 plasmid vector. when i screen using PCR i get the band of PCR product at an actual size of 1375 bp. but when i use double digest i do get...
30 January 2017 4,762 3 View
I have amplified 500 bp up- and downstream of my target gene and placed a KanR for selection between both 500bp fragments. Is that correct? Now, I want to use pGMT-t vector (Promega) as a...
27 January 2017 8,659 3 View
When determining titer of T7 phage by plaque assay, I get a gradient of plaques where one side of the plate is clearing and the other half of the plate is a lawn of bacteria. Anyone experience...
24 January 2017 267 4 View
I used Qiagen RNeasy Plant Mini Kit to isolate RNA from Turmeric Rhizome (6 months old) and used tertiary rhizomes as a sample for RNA isolation. I used two varieties (First Three columns...
21 January 2017 7,629 13 View
Hi! I'm trying to perform a ChIP assay on SH-SY5Y cell line. Can anyone suggest me which duo of antibodies and primers to use for a positive control of ChIP? Also has anybody an experience of a...
20 January 2017 7,511 2 View
I have three types of samples including liquid samples (water, milk), solid samples (feed, feces) and aerosol samples. After quantifying by qPCR, I have the copies number of each sample. However,...
19 January 2017 10,030 8 View
Hello (sorry for long text) I've tried the isolation in three different ways, in the second trial I changed some steps of first trial which are decribed by “ / ” after related step, in the third...
19 January 2017 6,695 8 View
Hi, How i can remove polysaccharides and polyphenols during the DNA extraction from Terminalia arjuna, i have tried various methods but unable to find DNA in purified form? I have tried many...
18 January 2017 7,891 2 View
Hi all, This seems to be a fundamental thing but I have this doubt. I have biological replicates of a timecourse experiment with a particular treatment. I wanted to know if I can combine both to...
17 January 2017 4,824 3 View
RNA profile for all my samples shows strange big peak at 5S position. Should I use these RNA samples for downstream experiments? Does anyone know if this is common with Staphylococcus...
17 January 2017 1,515 5 View
I need to make a 1% casein agar for a protease assay, but I'm finding it very difficult to get the casein to dissolve. The media needs to be at ~pH5.8. It is used in several published papers, but...
16 January 2017 7,725 1 View
Hi everyone! do you have any piece of advice about RNA extraction from microvescicles? I usually recover MVs from human monocytes supernatant. I tried different protocols, like trizol and columns...
16 January 2017 8,052 4 View
I am trying to prepare agar plates from the CHROMagar powder and the instructions say to mix 33g in 1L of water and bring to the boil at 100 degrees before autoclaving. This is difficult for me as...
16 January 2017 3,260 3 View
Hello, the issue I am facing is I would like to put a his tag on the N-terminal of my GPCR, but it has a signal peptide for the first 20 or so amino acids and I am unsure of how to get around this...
16 January 2017 6,450 3 View
I plan to use Lipofectamin 2000 to transfect human macrophage in vitro. Macrophage is not a easy target for tranfection and there are not many papers on tihs topic. Does someone has some...
13 January 2017 9,853 4 View
We are currently using the digestion solution below to digest cells and matrix grown within 2% agarose gels however large pieces of agarose still remain. Any suggestions on how to trouble-shoot or...
13 January 2017 1,153 3 View
Hello everyone! I'm starting a project involving phage display and the protocols largely sound like I can do my panning on the bench top, instead of in a biosafety cabinet. Do I need to pan...
12 January 2017 2,670 1 View
I want to see the community structure of the soil amended with biochar, however, I've got difficulty in extracting high DNA yield. My samples did not pass the quality control for next generation...
12 January 2017 6,208 18 View