I'm currently trying to transform top 10 E.coli cells that have streptomycin resistance with a ~5kb pET22b+ vector with ampicillin resistance in which I have ligated a ~1.7kb product but so far I have not obtained any successful colonies with the vector inside! I'm running out of ideas to try and fix this problem as I have tried different ratios for the ligations, warming the plates before transformation, leaving the plates as long as possible etc The two antibiotics that I am using are streptomycin and ampicillin, both of which are in the plates on which I place the transformed cells. In my most recent attempt to do this I doubled the amount of ampicillin on the plates as it is quite a weak antibiotic but absolutely nothing grew which leads me to believe the transformation did not work. Any advice/help would be greatly appreciated :-)
first of all, the postive control and negative control are very important! use any A+ vector(even empty pet22b+) to verify the transformation system and ampicillin has no problem on this vector.
if the competent of E.coli is self-made, the growth stage and the wash are very important. use ddH2O and discard the supernatant as thoroughly as possible.
1) insert quality - ligate insert to it self, ladder of products should be formed after 5' RT
2) Restriction sites - Nde I is popular but it is horrible for digestion/ligations
3) If you make insert by PCR, make sure there are 6 (more) bases between the termini
of product and restriction sites (I just put atatatatat)
4) recovery time - since streptomycin will inhibit protein production 1 hr recovery at least is mandatory
5) Test your cells with known plasmid
6) Make sure that plates are not too dry (it should take couple of minutes to adsorb
the recovery mix)
7) Never give up :)
I think the ligation is not likely to be the problem. Have you run any controls? For example have you tried transforming a complete plasmid (such as pUC19) into the cells? For the transformation you need to check the time constant of the electroporation is good - check with other people for the particular values you expect from your machine. For streptomycin you need to incubate for 1 hour at 37oC in SOC after transformation before plating to make sure your cells are resistant. Are you sure it is streptomycin as this is not the normal resistance for pET series vectors (normally ampicillin)?
If the transformation is OK then you can try gel purifying your restriction digest fragment for ligation. You should make sure your ratios are correct (3:1 insert to vector) and leave the ligation at 4oC overnight. DO NOT transform more than 2uL of the ligation reaction as this can cause the electroporation to short, killing the cells. If this still doesn't work then you can try a different vector assembly method such as Gibson assembly or golden gate cloning.
Hope that helps.
Ellis
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