Currently, we are trying to get a 3kb plasmid containing RFP constitutively expressed into P.a. We are following the protocol in this paper:
10 minutes preparation of electrocompetent P.a.
I will summarize the steps which we used as below:
1. Inoculate single colony in 6ml of LB overnight.
2. Distribute cell culture equally into 4 microcentrifuge tubes.
3. Centrifuge at 16000 x g and room temperature for 2min and discard supernatant.
4. Wash each tube of cell pellet with 1ml of sucrose solution and centrifuge. Repeat Step 4 twice.
5. Resuspend all the 4 cell pellets with a combined volume of 100ul of sucrose to produce 10^-9 viable cells.
1. Transfer 3ul or 500ng of purified DNA into 100ul of electrocompetent cells.
3. Transfer the mixture into pre-chilled cuvette.
4. Slot cuvette into BioRad Gene Pulser and pulse shock (2.5kV, 200Ω and 25µF). Add 1ml of LB at room temperature immediately into the cuvette. Transfer mixture into 1.5ml microcentrifuge tube and shake for 1 hr at 37oC.
5. Centrifuge at 16 000 x g and discard 900ul of supernatant. Resuspend cells in 100ul of residual medium.
6. Plate entire cell culture onto LB plate with suitable antibiotic (Chloramphenicol 20ug/ml). Incubate at 37oC overnight.
The only 2 steps which are slightly different from the paper are in bold:
- After the pulse shock, it takes us about 30 seconds (not immediately as suggested) before we add LB, as we need to move the sample into fume hood.
- We incubate the cells for 1 hour in 1.5 ml microcentrifuge tube, not glass tube.
The problem we encounter is that, after this 1-hour incubation step, the cell solution develops a transparent glue-like matrix which attach to all the cell pellets. We suspect that this might be biofilm, but have no idea which leads to its formation. So we always end up plating both the cells and this matrix on the plate. The next morning, we have indistinct colonies growing all over in all the plates, including negative control (cells without plasmids added). So it might be that this biofilm protects the cells from the antibiotics.
Have anybody else encountered this problem before, or had any suggestion on this?
Thank you very much in advance :-)