Hello all,
I have been trying to transform my plasmid ~10kb in comptent E.coli DH5a cells for the past one and a half months by heat shock method but haven't been able to get a single colony. I am doing transformation by the standard protocol i.e., by thawing competent cells (50ul) on ice in microfuge tubes, adding around 5ng of plasmid, incubating on ice for 30 mins and then giving a heat shock at 42°C for 90 seconds in a water bath (also tried giving heat shock for 40 seconds) and then putting cells on ice for 5 mins again. After that I added pre-warmed 1ml LB and kept them for 1hr in shaking incubator. Then spreading it on amp+ plates with a conc. of 100ug/ml.
I can't understand what am I doing wrong. I always prepare fresh ampicillin stock for my plates and made competents cells many times by CaCl2 method but never have been able to get colonies. Can someone guide me regarding this?
Hi Mudassar,
Can you specify what plasmid you want to transform
Your protocol to prepare competent cells by the CaCl2 method
and your preservation method (since there is "thawing" in your explanation)
@Jonathan Jonathan
I am afraid I can't specify the plasmid
The competent cells are prepared from the secondary culture when its O.D600 reaches at 0.4. The culture is centrifuged to get pallet which is then washed in 0.5 M CaCl2 0.5 MgCl2 solution and then preserved in 0.5M Cacl2 with 15% glycerol and stored at -80°C after treatment with liquid nitrogen. Our lab has been using this protocol without any problem.
It looks like there is something toxic on your plasmid. Also, be careful of the antibiotic resistance, not all plasmids contain AmpR cassette (i.e. use the right plates!)
Thankyou all for your valuable suggestions. I tried electroporation and it worked.
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