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Questions related from Gen Gi
I am getting zero DNA yield after using qiagen purification columns. I finally traced the problem to NEBuffer 3.1, but pH doesn't seem to the cause. Essentially, I observe: 3 ug of DNA in 50 uL...
05 March 2024 5,835 6 View
I'm doing CRISPRi with dead spCas9 on mouse cells, and normally we do the following for gRNA design: 1. 20 nt spacer 2. Extra G on the 5' to enhance expression from hU6 promoter. Final product:...
26 February 2024 4,363 3 View
I need to find a mutant allele of my protein that is defective for homo-oligomerization, but can still bind a separate protein (heterodimer formation). I think I will do alanine scanning...
08 January 2023 2,778 5 View
A lot of answers/literature online say that T4 ligation of >3 fragments is very difficult/occurs at low efficiency. However, I also see reports/protocols everywhere of Golden Gate being used to...
20 September 2021 7,112 2 View
I have been using NEB Hifi Gibson assembly for a couple years now and I've been quite happy with it. I regularly make plasmid constructs with 4-8 fragments, and always >1/4 of the colonies are...
10 July 2021 1,469 2 View
To generate stable mammalian cell lines, I am doing a kill curve to find the optimal concentration of antibiotic for selection. The problem I have is that, even with a very high level of...
08 March 2021 8,735 2 View
I must put 50 kb plasmid into ecoli strain. I think 50 kb is too big for heat shock with ecoli. Is that correct? Will electroporation work? Or do I need to use cos sites and lambda phage...
23 January 2021 8,836 3 View
I received several strains of bacteria from another lab far away. However, one of the samples appears to have high fungal contamination. When I grow it in broth, I see lots of fungal...
04 January 2021 2,964 16 View
I made a new bacterial strain with an unmarked deletion using 2 step homologous recombination (with sacB counterselection). The full protocol is given in the link at the bottom. The protocol...
02 January 2021 1,799 4 View
Sometimes my western blots have very uniform/even background, other times they're very uneven and patchy like in the attached image. Why is this? I thought my blots were drying out during...
06 December 2020 8,941 5 View
In the past, I've always used Licor's old blocking buffer that included sodium azide. I would dilute my antibodies in the buffer, stain, re-collect the dilution, and re-use it a few times. The...
29 November 2020 6,034 3 View
I have a low copy plasmid (ori=pBR322) and I wanted change it to high copy (like ori=pUC). This paper seemed to say that only one mutation was required to increase the copy number: High copy...
14 November 2020 7,472 4 View
For western blots, I normally stain with 10 mL buffer with 1:1,000 dilution of my specific antibody. Recently, there is an equipment change in my lab and 10 mL isn't enough volume to cover the...
28 October 2020 2,957 7 View
I must concentrate and purify a lot of dirty lysate and secreted protein samples. I just want to run them on protein gels for western blots. I'm trying to decide the best method of...
11 September 2020 3,107 8 View
I have some plasmids with sacB. Ecoli carrying these plasmids seems slow even on standard LB without sucrose. I sequenced the plasmids and everything seems normal. Normally I have nice colonies...
23 August 2020 3,434 3 View
Normally, ecoli miniprep cultures are grown for around 12-18 hours at 37C. However, due to my schedule, I won't be able to harvest the cultures for >24 hours. I think >24 hours is too long to...
18 August 2020 8,011 6 View
Hi, I was just given a plasmid for sacB counterselection in mycobacteria. I sequenced this plasmid and I was surprised to find there is a mutation in the signal peptide of the sacB gene. The...
28 July 2020 3,016 3 View
I'm trying to perform a BCA assay on some clarified lysate, but the samples appear to become brown, not purple, and the OD562 absorbance is very high. What could cause this? The brown is very...
11 July 2020 7,146 3 View
I am trying to purify a plasma membrane protein from ecoli. I think the standard procedure for membrane protein extraction is like this: Lyse Low speed spin to clarify (eg, 5k xg for 20...
03 July 2020 6,112 3 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 744 8 View
I am working with an unusual bacteria that is very hard to lyse. I want to extract protein and RNA from this bacteria. I need to determine the optimal lysis conditions for this bacteria, but I am...
28 June 2020 5,111 0 View
I did two PCRs One band was very, very faint while the other was very strong. Both were made from one master mix (without primers) Both used the same template (bacterial gDNA) Both were put in...
27 June 2020 1,778 11 View
I recently saw the paper: "The +4G Site in Kozak Consensus Is Not Related to the Efficiency of Translation Initiation" The +4G Site in Kozak Consensus Is Not Related to the Effici... Which...
03 June 2020 6,115 0 View
For bacteria like ecoli. For regulatory elements that control transcription, how common is it for them to overlap with the sequence of the upstream gene? I know I can find examples in the...
29 May 2020 1,933 0 View
For bacterial promoters, everything I read says bacterial promoters are -35/-10 motifs upstream of the TSS, and perhaps UP elements (~ -50 of TSS). So if I want a promoter sequence for my gene,...
09 March 2020 1,010 7 View
I want to extract membrane+cytosolic proteins for western blots. I'm working with hardy bacteria, so I have to bead beat for full lysis. During bead beating, the manufacturer protocol recommends...
04 March 2020 4,849 20 View
I'm extracting protein from highly hazardous bacterial samples. For the safe of safety, people commonly boil this bacteria prior to full lysis when doing protein extraction. Because the bacteria...
03 March 2020 901 8 View
Many microbiology processes involve: 1. Growing a starter culture to high density 2. Back diluting into fresh media by some dilution factor, eg, 1:100 3. Growing to OD X (eg, OD=0.5) 4. Harvesting...
17 January 2020 982 10 View
Many microcentrifuge tubes are designated as both "RNase-free" and "non-sterile." What does this mean exactly? I can't imagine something could truly be both RNase-free and non-sterile. Is it...
16 January 2020 9,004 3 View
Does anyone have/know any data on how the volume of recovery media affects things for electroporation of bacteria? I see large ranges. For electroporating, eg, 200 uL concentrated bacteria,...
13 January 2020 4,999 3 View
I'm extracting RNA from bacteria and I only get a yield ~20% of the time. I can take one culture, split it into multiple tubes, prep them in parallel, and only one tube will give me RNA. I...
11 January 2020 9,738 2 View
So I'm using TRIzol for RNA extraction, but suddenly I'm getting no pellet during the isopropanol step *even though I added glycogen.* A few weeks ago I: 1. Took a large quantity of bacteria 2....
06 January 2020 8,500 5 View
I'm using Ambion's TURBO rDNase and I think I'm seeing degradation of my RNA. Why is that? Lane 1 = untreated RNA extract. 23s/16s intensity ratio = ~1.8 Lane 2 = ~0.5 ug of RNA, 37C for 30 min....
01 January 2020 3,942 2 View
I'm using TRIzol+bead beating to extract RNA from a gram positive bacteria, but I'm getting very low yields (
03 December 2019 9,772 3 View
I've been using LN2 for snap freezing a lot, and the effect is near instant. One second my samples are room temperature, the next second they're frozen solid. I've read dry ice + 100% ethanol...
27 November 2019 1,720 5 View
For TURBO DNase, is buying the full kit that uses bead-mediated removal of the DNase worth it? Or can I just buy the TURBO DNase itself and use heat inactivation? I'm mainly interested in using...
25 November 2019 6,354 1 View
I want to make stocks of electrocompetent bacteria, but, due to safety concerns, I may not be able to snap freeze using liquid nitrogen or dry ice. Can I just stick them in the -80? How much...
24 November 2019 6,671 3 View
We work a lot with an enda+/endonuclease+ strain of ecoli (stbl3), so for minipreps we just do the PB wash step and things work fine. However, we sometimes have trouble with low quality DNA from...
21 November 2019 9,498 1 View
I transformed a bacteria with an integrating plasmid. I grew up colonies in broth and then did colony pcr to genotype them. I did two pcrs: 1st pcr: Should only give 1 kb band from transformed...
01 January 1970 7,862 11 View
