No colonies after transformation! - (Mar/09/2011 )
I am having alot of difficulties in transforming a construct purchased from a company. The details are as follows.
Vector: psiHIV-mH1 (HIV based) 8870bp
insert size: shRNA 63bp
plasmid: 8ul (10ng)
Competent cells DH10B: 20ul
I added the circular plasmid to the competent cells (after thawing the cells on ice for 5mins) and allowed the plasmid - competent cells on ice for 30mins. After which I carried out the electroporation method. After which it was returned back to ice for 5mins and later the competent cells were flushed out using 500ul LB. The LB-competent cells mix were allowed to incubate for an hour and 10mins at 37C for recovery.
Plate 1: 10ul of the mix was used for streak plate
Plate 2: 15ul for spread plate
Plate 3: the remaining LB-competent cells was spun down at 6000rpm for 5mins and re-suspended in 100ul. Of which 50ul was used to make spread plate. (I carried out plate no3 as suggested by a colleague)
The plates were LB-Amp with Amp concentration of 100ug/ml. The plates were left in the incubator overnight at 37C. The next day morning I found there were no colonies.
Previously I carried out the heatshock method which also resulted in no colonies.
50s at 42C
Afterwards on ice for 2mins.
Recovery at 37 for 50~60 mins.
Iv also performed the transformation step at a lowered Amp concentration of 10ug/ml which results in bacterial growth with no plasmid vector at the end of miniprep. I believe the competent cells are efficient and works well for other transformations performed by my mentor.
At this point I can use any help I can get!
Transforming competent E. coli with a circular plasmid should produce lawns -- if your cells are indeed competent, your plasmid replicates in E. coli, the amp gene is expressed in E. coli, and your insert isn't toxic to the cells.
As Homebrew said, since you are transforming a plasmid into competent cell, you should get lawns of Ecoli... to many cells is the problem.
In this experiment there are only a few variable
- competent cells
First check the easy things
1 - are you using the correct selection LB plates? How much antibiotics are you using?
2- are your cells really competent?
3- do you have DNA in your tube?
4- is the electroporator working?
Can you find a plasmid with AmpR that you can use to test the competency of your cells?
LB should be okay. BUt I prefer using SOC medium for cell recovery.
How exactly are you plating the recovered cells? Do you plate the recovery medium + cells directly onto selection plates?