Problems with Transformation / DH5-alpha / Plasmid DNA ??!!
Raghavendra V
Wednesday, 16 January 2008 16:10 UTC
We have cloned a gene, and transformed it into DH5-alpha E.coli cells with suitable antibiotic (of course in the right concentration). The problem is, after incubating O/N at 37 deg C, we notice very tiny colonies on the LB-agar plate, and with great difficulty if we are able to locate, and inoculate a single colony into LB for Miniprep, at the end of the procedure we do not find any plasmid DNA!! (on 1% TAE agarose gel!!) Can any of you give a solution for this problem?!
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I dont know what kind of construct you are making, but consider the scenario in which the construct is toxic to the cell. It will cause reduced growth, and curing of the plasmid. Sometimes this can be circumvented using a plasmid backbone that is maintained at lower copy, or using a strain that maintains plasmids at a lower copy number (such as TG90 for instance).
Good luck! -
What controls do you use? Because that could be the problem
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I am also having trouble with my transformations. When I started cloning my initial projects were successful, but recently I have not been able to produce colonies. If any are produced they are empty.
From this forum I have decided to set up a control with uncut vector to check my cells.
Is there any thing else that no background would indicate. I am confident in my ligation set up, if anything I am worried that the vector is not cut properly, but this should lead to high background, right?
By background I mean colonies transformed with empty ligated vector.
I am using highly competent dh5alpha cells and pMES vector, and the Roche rapid DNA ligation kit.
Any input would greatly be appreciated.
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dear raghvendra
the problem be the antibiotic you used,so please do check with the conc. or some other antibiotic
thank you
shubhi
nccs pune -
now i start using topo ta cloning, i also facing problem in transformation.even in control i didn’t get colonies.
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If the empty vector doesn’t give any colonies either, have you tried using a different E. coli strain, like HB101?
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Anonymous
hi
u must see to tat the incubation time does not exceed the correct time limit.the tiny colonies u get are the satelite colonies which grow due to lack of antibiotic in he circular zone surrounding the desired colony..u jus have to keep a check on the time of incubation..
and abt the no plasmid problem,hav no idea.
act even i have a similar prob where my insert is not gettin ligated into the plasmid instead self ligattion occurs.. -
Hi raghavendra
There can be no of problems.
- Check out the ligation step do it carefully.
- Give heat shock properly.Before starting miniprep repeat first step no of times ie take the culture and settle down the cells then throw the supernatant then again take the culture and centrifuge it uptil you get good pellet
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Dear All, we are currently facing exactly the same problem which was discussed in this forum, i.e., tiny colonies and at the end we have empty (no plasmid) colonies… We are trying to clone a gene (2.3 kb) into a lentiviral vector (11.2kb). In brief, we cut our insert from a TOPO-vector, purify a 2.3 kb through agarose gel (Qiaquick gel extraction kit, cat. # 28704, Qiagen). Cut our vector with the same enzymes (double cut) and purify the vector through agarose gel. We control really each step during our preparations, i.e., after each enzymatic digestion and agarose gel purification , we measure OD and run an aliquotes on agarose gel and then do ligations. We use T4 DNA Ligase from Fermentas. We tried several molar ratios of vector:insert, from 1:1 to 1:5, but no success! If we use unpurified vector and unpurified insert for the ligation we get hundreds of colonies and they grow well. I would greatly appreciate any useful suggestions!
Raghavendra V, I was wondering if you could menawhile solve your problems with tiny colonies and if could share your experience with us. Many thanks in advance! -
hi,
i have seen a similar kind of problem.might be your cDNA is causing instability to the plasmid. try using a strain, STBL2 from GIBCO, which helps alleviate the problem.
aparna
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