I used the following procedure to transform DH10Bac E. coli.
I wanted to know why my experiment failed.
Exp. Procedure:
1. Thaw 25ul DH10Bac competent cells on the ice for each transformation.
2. Add pFastBac™ construct: 1 ng (1 μL) plasmid DNA to the DH10Ba competent cells and mix gently.
3. Incubate cells on ice for 30 minutes.
4. Heat-shock the cells for 45 seconds at 70°C without shaking.
5. Immediately transfer the tubes to ice and chill for 3 minutes.
6. Add 900 μL of room temperature LB.
7. Shake tubes at 37°C at 150 rpm for 4 hours.
8. Prepare 10-fold serial dilutions of the cells with LB Medium. Plate 100 μL of each dilution on an LB agar plate containing (50 μg/mL kanamycin, 7 μg/mL gentamycin, 10 μg/mL tetracycline, 100 μg/mL X-gel, 40 μg/mL IPTG)
9. Incubate plates for 48 hours at 37°C.
10. Pick white colonies for analysis.
11. Analyzing recombinant bacmid DNA by colony PCR.
As you can see, the result of DNA electrophoresis I can't detect the bacmid.
The primer I used tends to self-anneal.
Primer sequence:
pUC/M13 Forward
5′-CCCAGTCACGACGTTGTAAAACG-3′ pUC/M13
pUC/M13 Reverse
5′-AGCGGATAACAATTTCACACAGG-3′
Hello,
Did you test your primers on the pure plasmid beforehand? first look for amplification, maybe there is a problem in the colony PCR.
Why did you pick up the white colonies, did you do insertional knockout of the B-gal (lacZ) gene? If the gene is intact you should rather have blue colonies.
It is not the transformation that is a failure !!
On the contrary it is a success !!!
Indeed you get blue colonies or white bacteria. So transformation is OKay !
The problem therefore comes from before the bacteria transformation (ligation ...)
or come from after the bacteria transformation (colony-PCR ...)
To start, follow Sofiane Benyamina's excellent advice : Did you test your primers on the pure plasmid beforehand ?
Then I find that it has few white colonies compared to the number of blue colonies.
But this may be normal, it all depends on what you did before this transformation step:
What did you do before transforming the DH10Bac E. coli ?
Did you use the pFastBac™ vector as is, alone ?
or did you do a ligation beforehand, with this pFastBac™ vector and an insert ?
If it is a ligation you did before transformation, then details should be given concerning the type of insert and how did you prepare the vector ?
The insert : is it a PCR product, a DNA fragments, something else ...???
What are the expected sizes of the inserts, etc...
The vector : how did you prepare the vector ?
Did you do a double digestion with 2 different restriction enzyme? Did you purify the vector after the digestion, in order to remove the small fragment from the multiple cloning site ? It must be done to prevent from religating itself and with the vector, this would explain the high proportion of blue colonies...
If we are in the hypothesis of a ligation with a PCR product : have you properly purified your PCR product before the ligation ? This is to remove the not incorporated primer-dimers that could compete with the insert (the PCR amplicon) for ligation within the vector....
The colony-PCR can be tricky. When the colony is taken from the Petri dish, care should be taken not to poke into the agar. The agar contains taq-polymerase inhibitors...
In summary, problems can arise at each step. That's why you need to give more details ...
Good luck !
- Badges
- Science topic