Hi researchers!
I made my own E. coli BL21 (DE3) competent cells with CCMB80 buffer working with an 0,52 OD. And the day after I performed my transformation protocol in this way:
1- 20 ng of plasmid DNA (my control) in 100 uL of competent cells (stored in 10% of CCMB80).
2- 20 ng of plasmid DNA (the plasmid I'm looking to insert after a MiniPrep) in 100 uL of competent cells.
3- 400 ng of plasmid DNA (the plasmid I'm looking to insert after a MiniPrep) in 100 uL of competent cells.
(following the protocol...)
Incubated on ice 30 min, heat shock at 42 C during 30-40 seconds, incubated on ice 2-3 min and then added 250 uL of prewarmed LB at 37 C. And let them recover during 1-1:30 hour. After that I served 100 uL, 100 uL and centrifugate and plated the pellet from 1 sample, that's the same I did with the other two samples. Plated in LB + Amp 100 ug/uL.
And incubated at 37 C during 14-16 hours.
RESULT:
1 - successful transformation (my control)
2 - failed
3 - failed
After that, I made another experiment using another plasmid (from the same sample being a replica).
4- 50 ng of plasmid DNA (replica 2) in 50 uL of competent cells.
5- 50 ng of plasmid DNA (replica 3) in 50 uL of competent cells.
6- 50 ng of plasmid DNA (replica 4) in 50 uL of competent cells.
None of 4, 5, or 6 showed any growth.
Is the plasmid e.coli compatible that you want to insert.. since you are transforming pure circuler plasmid.. not ligated DNA, there should not have any problem in transformation in stored competent cells... But i would suggest to prepare fresh competent cells and repeat the experiment
I would recommend making slight changes in your protocol. Give the heat-shock for a minimum of 60secs (could be extended to 90secs max if required) and then keep on ice for 2-3mins.
Nextly, add 200ul of LB, incubate for 1-1.5hrs & then plate around 70-80ul.
Or
Add 1ml of LB media, incubate for 1.5hrs, centrifuge at 4000rpm for 5mins. Discard most of the sup carefully such that around 100-150ul remain. Mix it gently by pipetting and plate around 70ul.
Try not to use competent cells that have been stored for a while.
Hi
As per your problem, I suggest first you have to incubate the competent cells in ice for 20min and then follow your protocol. After heat shock step, you have to incubated the sample in ice for 20-30min and then add pre- warmed LB media and incubate this sample at 37c with gentle shaking and then follow your protocol.
If you wouldn't get positive results after following this protocol then definitely you have to standardize the competent cells preparation protocol.
When you had success in the control experiment (#1) did you get hundreds of colonies or hundreds of thousands? The plate should have been nearly confluent with colonies.
I think the most likely answer is that your competent cells just are not very competent at all.Minor tweaks like changed the incubation time on ice or the heat shock time might make a small difference (2-3 fold at most) but if you are getting nothing then the problem is much different than a minor tweak in protocol.
Were the other plasmids also uncut, or was this cut and ligated?
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