I am trying to clone an 8 KB fragment to pFLAG-CTC vector. The vector map of the pFLAG-CTC vector is attached herewith. The issue I am facing is that after ligation reaction, and transformation not single colonies I find either in ligation or vector control. I am using E.coli DHB10 cells. And after transformation, I incubate the cell suspension in 37 Degree celcius.
The Protocol is as under:
1. I take 40 microliters of competenet cells of E. coli DHB10 cells in an cold electroporation cuvet.
2. I add 3 microliters of ligation mix or vector control to the lectroporation cuvet.
3. Perform electroporation at 25 microFaraday capacitance, 2500 volts and pulse controller unit to 200ohms.
4. I Immediately add 1 ml of SOC broth to the cuvet and mix.
5. Transfer the cell suspension to 1.5 ml of centrifuge tube and incubate in shaker incubator at 37 degree celcius for 3 hours.
6. Centrifuge the cell suspension at 4000 g for 3 minutes.
7. Discard the supernatent.
8. I resuspend the cell suspension in 100 microliters of LB broth and pour into two LB Agar plates containing 100micrograms of Ampicillin per ml Media..
9. Incubate the plates in 37 degree celcius for overnight.
If i understand correctly, you did transformation but couldn't get any colonies in both ligation and control vector. If there is no colonies in even the control vector, that means either the competent cells are problematic or the transformation protocol is problematic.
1) try to check if you competent cells are still viable, just spread some on media without antibiotic to see if they are viable.
2) Actually, after step 5, you can directly plate the cells at about 200-300ul per plate.
3) Try check your antibiotic. Does you cell or plasmid have resistance to ampicillin?
hi
I partly agree with the opinion of Wong " check competent cell viability before ad after ecletroporation by LB-Agar" , but you can do it :
1- Make sure to use "electrocompetent cells "
2- use 1mm cuvett , then use salt free or clean up ligation reaction , cells+ligation let 5 min on ice
3- check optimal voltage and mode of instrument " high voltage for long time cause cell death "
4- Pipetting it gently ( 1or 2 times)
5- 50 RPM / 37 c / 1 hour
finally if your competent cells viable before electroporation and death after electro shock you can do :
1- change instrument parameter
2- use chemical competent cells and done heat shock method
good luck
Hello,
I think adding the ligation reaction directly to electrocomptent cells might be causing your problem I suggest you either do an ethanol precipitation or use a cleanup kit for your ligation rxn before transforming it. Also make sure you are using 2mm cuvettes as the settings you specified are usually used for this cuvettes. Finally, your are not recovering your cells properly you need to transfer them to a round bottom 15ml falcon tube for recovery because in the eppies they neither get proper oxygen nor shaking (it is true people use eppies for convenience however that is used for simpler transformations where the number of transformants is high). I suggest you recover for 1 hour then plate half of your transformation then incubate for 2 addition hours then plate the rest.
Best of luck
Hanna
Have you confirmed the presence of your insert in the construct and the correct frame? if all is intact, you may revisit your transformation. Why not try electroporation rather than using chemically competent cells.
Sounds like your cells are dead. Possible issues:
Cells are not viable before transformation. Test by streaking a few on plain LB and look for growth.
Cells killed during transformation. Test by ruling out the above. Check the settings on your equipment. If you hear a "pop" noise or see sparks, you've killed your cells.
Mistake in LB + amp plates. Double check that you added the correct antibiotic in the correct concentration. Too much antibiotic can inhibit growth.
Good luck!
how certain are you that cells are alive? due the fact not even the control is growing, also, never used the plasmid, what selection cassette does it have?; do you "activate" the cells on a broth before use? sometimes, if are freshly thawed, many bacteria are dead or die within a stressful process such as transformation.
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