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No colonies after transformation!


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#1 junks18

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Posted 09 March 2011 - 06:51 PM

Hi,

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

Transformation step:
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!

~Junks~

#2 HomeBrew

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Posted 09 March 2011 - 08:00 PM

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.

#3 perneseblue

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Posted 09 March 2011 - 08:54 PM

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
- plasmid
-equipment
-selection plates
-protocol

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?
May your PCR products be long, your protocols short and your boss on holiday




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