We have now attempted several times to transform our pCS1966 plasmid 5.4kb(approx.), containing an insert. The pCS1966 contains a erythromycin resistance and an orotate transporter. We have tried to use the ordinary heat shock method, but results were negative. We tried a thermo scientific transformation kit as well, which did not succeed either. With the thermo scientific protocol the positive controle did not show colony's either, meaning that the plasmid itself without insert was not transformed either. Does anyone have any suggestions as to how to continue. We are doing a 2 steps ligation, we have 2 fragments, a and b, A is 700bp, B is 1000bp (approx) We have stared with fragment B as the insert.
Are you saying that you have been unable to transform the positive control (pCS1966 without insert) in both cases?
Have you got the resources to try with electrocompetence? It's a highly efficient method, although the cells require some previous treatment (sorry but I don't have the protocol nearby right now). Also, what the previous answer says: check the positive control with the empty vector.
What you mean with results were negative, no colonies appeared or none of the colonies had the plasmid??
The creators of pCS1966 use E .coli strain ABLE-C, which lowers the copy number of ColE1 derivatives, to reduce cloning problems due to toxicity of cloned fragments in E. coli. Are you using this strain ? If not, this might help.
http://www.malariaresearch.eu
Using erythromycin for selection against E.coli seems tricky sometimes. It may happen that the promoter is inducible and you may need to expose the cells at lower concentration of erythromycin for induction first. It is better to try with some other vector, if it is not so stringent to use pCS1966 for your purpose.
Have you tried the method of electrocompetent cells?, this method has a high efficiency compared to thermal shock
check the competent cells, Are the cells able to grow on agar plate without antibiotics.
Only the thermo scientific protocol had a failed positive control. Heatshock positive control was succesful, meaning the plasmid did go in and gave colonies. We use e.coli strain dh5alpha and yes,we do have an electroporator. We have to use this plasmid but not this strain. We will look for a eporator protocol and see if the other e.coli strain is in stock as well. Thank you all !
As Arpan said Ery in Ecoli is tricky, most of E. coli strains are resistant to high concentrations and the Ery genes used in these kind of plasmids are from gram positive bacteria. Some labs used special E. coli strains. To help you if would be great if you gave us more details, concentration of Ery used, results (no colonies, no plasmid, etc...)
The used concentration of erythromycin was 150ug/ml. We have used 2 different methods. The first was the normal heat shock method, where we used 2 controls. The positive(cells+original plasmid) control was succesfull meaning they did show colonies. The negative control was the plain bacteria and that one didn't grow. The modified plasmid however did not transform into E.coli strain DH5alpha
The second time we used a thermo scientific protocol, in which competent cells were made and the transformation should have been done. The same controls were taken as in the experiment before and positive control was negative. So the original plasmid was not transformed into E.coli DH5alpha and the modified plasmids were not transformed either. We will make a dilution for several concentrations of erythromycin to see in which one they will grow. Thank for the tips everyone!
Did you chech the presence of the plasmid in the positive cotrol?? 150 ug/ml should be a good concentration for selection as shown in the negative control. peharps the efficiency of transformation was not good enough for the ligation??
We are certain that the plasmid was in the positive control. The ligation is not the issue, we were able to check the ligation using PCR with specific primers to our region of interest, so the ligation is correct. The only part so far not succeeding is the transformation of this ligation. We need to create more plasmids, so we can isolate these and then insert fragment A(700bp approximately).
We are first going to try to make a dilution of erythromycin to see if this might have been the cause. We will ask if there is the E.coli ABLE-C strain available in our university, to see if this will help.
We have reattempted it 3 times now. Using heat shock, the kit, and electroporation. All the controls were as they should be, but both the heatsock and the kit did give colonies. The electroporation however did give kolonies, but also a lot of background. The protocol does require 1.0x10^11 cells per cuvet. Is it possible that this large amount of cells caused a background on the erythromycin plates?
To be sure about the kolonies, we made a subculture and we are going to do a colony PCR of a possible successful transformation as wel as a PCR of the control, to be sure it is the right plasmid/bacteria.
Yes it is possible that you get a background of false positives if you have many colonies per plate. you can either plate more dilutions (next time) or do what you did subcultures to identify positive ones