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Historically, I've had no difficulty with TOPO TA cloning, but I have had great difficulty recently with very inefficient cloning reactions - many transformants, but almost none contain inserts.
Any number of approaches that I've taken recently have resulted in total absence of insert. The only similarities among the 6 primer sets (different targets) that I am using are the restriction enzymes on the 5' termini of the oligos. I am aware that 5' bases can impact cloning efficiency (Peng et al 2007; Liu et al 2007). Is it possible that certain 5' termini can reduce transformation efficiency to 0?
Below are details of my experiments and a list of the troubleshooting approaches we have taken.
PCR from genomic DNA isolated via phenol/chloroform and ethanol precipitation (no proteinase K or RNAse digestion)
~6 primer sets against different targets, all with the same restriction sites on 5' termini; forward primer have a 5' NdeI site (CATATG); reverse primers have a 5' KpnI site (GGTACC)
Troubleshooting experiments and observations:
(1) Control TOPO TA reactions with Invitrogen supplied pUC and primers yield =>90% cloning efficiency (direct colony PCR with T7/M13R) and many colonies
(2) Control TOPO TA reactions work for 2 different TOPO kits (same competent cells)
(3) Taq polymerase choice (NEB or Fermentas) makes no difference. Both should yield A-overhangs.
(4) All primer sets yield the same difficulty.
(5) Blue white selection - lots of blue colonies and a few (<0%) white colonies. White colonies do not include inserts.
(6) Direct colony PCR of transformants with T7/M13R yields a small fragment - this may be the ~190 bp amplified polylinker but could include something else small.
(7) Gel extraction didn't solve the problem, but # of transformants very low. Recircularization of the plasmid may have been the problem.
(8) TOPO TA reactions of fresh PCR product and product stored several days in refrigerator yield equivalent results.
(9) PCR cleanup yielded transformants, ~95% of which are blue in blue/white analysis with only a few white colonies (each containing insert as determined by direct colony PCR)
I have cloned and expressed my protein in BL21DE3 cells using pGEX-4T2 vector and have succesfully purified the recombinant protein using an affinity column (glutathione). When I tried cleaving the GST tag with the RECOMT thrombin cleavage kit (sigma), I didn´t see any cleavage of the tag from the protein when looking at the gel. I have been facing this problem for some time and have not yet been able to obtain protein without the tag. I have carried out the reaction at around 25 degrees, as directed by the kit, for 24 hrs but with no result.
Can anyone please tell me if you have used another technique or kit for performing GST tag cleavage?
I eluted my RNA after the RPE wash during the RNeasy kit and realized that my sample has carried over a lot of ethanol. How do I remove it? Should I re-precipitate or just let the ethanol evaporate? How do I re-precipitate to get rid of the ethanol in my final sample without losing too much yield.
My gene of interest cloned in pET28a with C-terminal His tag is expressed in arctic express E.coli strain. Protein is in soluble form and using GE fast flow Ni-NTA beads for purification. My protein of interest is getting eluted in flow through. Please help me in this regard
I want to transfect genes after ligation of 4 subloning gene. I have done PCR on genes, affect restriction enzyme on PCR products and vectors, cleaned up and then ligation using T4 ligase. I tried transfect the genes to TOP10 CaCl2 competent cells but I got noting (No colony on AMP+ plate)
Protocol for competent was:
Overnight culture of TOP10 stock in 10 ml LB, then 80 microliter of culture to 6 ml LB for 8 hr,, then centrifuge 8000 g 5 min, resuspend, add 1 ml CaCl2 (0.1 M) cold, 30 min on ice, centrifuge 8000 g 3 min, discard supernatant, 0.670 ml CaCl2 (0.1 M), 30 min on ice, centrifuge 8000 g 3 min, discard supernatant, 0.330 ml CaCl2 (0.1 M), resuspend
Protocol for transform:
8 microliter of ligation + 50 microliter TOP10 competent, mix, 30 min on ice, 2 min on 42 C (thermoblock), then 1o min on ice, then added 1 ml LB with out antibiotic in 37 C, then centrifuge 8000 g for 5 min and then discard 800 microliter of supernatant and transfer of resuspend to LB agar + 100 microgram/ml ampicilin and incubate for 24 hr
And I got nothing
In the protocols which points are critical?
Is there any beter protocol?
Can I transfect by electroporation? How it works? There is a deteailed protocol?
(using ependorf electroporator)
I have a problem with transformation. I performed ligation at 4 degrees and stored at 4 degrees.
Vector (Digested with EcoRI and SmaI) (9.8kb)
Insert (Digested with EcoRI and SmaI)(1.8kb)
After digestion I performed gel extraction or elution, yield was very good on gel, by using 1 microliter. I proceeded to ligation, I used T4 DNA ligase, NEB nad 10X NEB buffer.
I did transformation with freshly prepared E.coli MC1061 competent cells. The problem is I did not get any colonies on the plate after ligation but I have a good number of colonies on positive control. (I think my competent cells are good). I dont understand, where exactly the problem is? Does anyone know where I might be going wrong? Are my ligation conditions ok?
I am using the CaCl2 method for competent cell preparation but recently I am facing low efficiency of transformation in new competent cells despite several tries. I always test them by doing transformation of approximately 1 ug of plasmid DNA that used to fill the plate but now I am only observing approximately 20 to 30 colonies each time!
My clone (insert + vector) is 11kb long, I already cloned it in TOP 10; Stbl3, XL 10 Gold, NEB 10 beta, SURE and ABLE K. All by quimical transformations. My next experiment will be cloned in electrocompetent TOP 10. My problem isn't the transformation, because after the colonies screening my insert (~ 1000pb) is there, but when I place them on LB liquid or with agar to grow up and make the miniprep, it disappears. Seems that the bacteria lose or reinsert the insert.