Originally Posted by ismawanto
Currently we are working with pWATERGATE plasmid (18050 bp). We have transform this plasmid into ccdB survival E. coli (Invitrogen) by using heat-shock method, then spread it on LB plate containing 10 microgram/ml Spectinomycin. We trasferred the colony into LB media containing 10 microgram/ml. When we isolated the plasmid from E. coli culture, we got low concentration of plasmid. We use alkaline lysis with SDS method (Sambrook and Russell).
pWATERGATE is a Gateway system vector (Invitrogen). All of those plasmids are low-copy
so you will get low yield (<100 ng/uL in a miniprep) unless you grow it in a larger batch and purify it from more culture. For low-copy plasmids and mini-preps, I usually start with 10 mL to 15 mL culture, but a midi-prep would be better.
In order to confirm this plasmid, we digested this plasmid by using SacI and XhoI enzymes (double digestion). According to the restriction map, this plasmid only has one restriction site for those two enzymes. So after digestion it would produce 2 bands (16.3 kb and 1.7 kb). But for our case, after digestion it only produced single band (about 9.5 kb). Does it mean that we have isolated the incorrect plasmid. |
Thank you in advance.
What size does the undigested plasmid run?
This is a hairpin vector for RNAi. Typically RNAi vectors are prone to recombine, especially I imagine huge bulky ones like this, so you can't keep them in culture too long. How long do you grow your culture before you perform the plasmid prep (I would recommend <18 hours if possible)? Have you tried screening several different colonies after transformation or do you only subculture one colony? Have you tried inoculating culture directly from a glycerol stock?