Mutagenesis Help (plasmid isolation & sequencing stages) - (Aug/24/2006 )
I am an undergrad trying to get through a small mutagenesis research project.
Trying to do a point mutation on an enzyme, I have thus far only mutated the plasmid using PCR. I ran an agarose gel on the product and saw a band at the right weight; however, I've been having problems with transformation. I follow the Quikchange protocol (Stratagene) all the way through, but when I try to extract plasmid from transformed 3 mL cultures, nothing shows up on the gel. I've also done EtBr spot tests to show very little (6.25 ng/uL) plasmid concentration.
With this in mind, my first question is: what do I do to get more plasmid?
The next step (as I've understood it) is to sequence the plasmid to make sure it's what I want. This step brings further questions. Namely: how do I prepare the plasmid for sequencing (I understand that we have a sequencer)? Now, I know how sequencing works in theory, mind you, but all I've read describes manual sequencing. My other question is: can I use the same primer for the PCR reaction (I guess this assumes the dideoxyNT termination sequencing rxn) that I used to mutate the wild-type plasmid? If not, does anyone have primer desgin tips?
hmm... a problem with plasmid yields...
There are several things that you can do which comes to mind
- Increase yields of plasmid by changing quickchange amplification conditions
- pool several (8 - 16) reactions together and make a concentrated mix for ligation
- assuming that your competant cells are okay, you could purchase some ultracompetent cells from invitrogene like genehog... which take up plasmids really really well, a thousand time better then XL1 blue. But cost blood.
I do find it odd that you could not extract any plasmid from the positive colonies that were obtained from the transformation reaction. If there was no contamination, then all colonies on the ampicilin selection plates,should contain some plasmid or another. The cells aren't naturally Ampicilin resistent.
As for the sequencing, you can go about it in two ways,
if your lab has the big dye terminator kit, you can do the Sanger reaction in house and then send off the finished product to be sequenced at a sequencing centre.
alternatively if the sequencing centre has the appropriate primers for your plasmid, y ou can pay extra and send the plasmid to the centre and they do everything.
As for preparing your plasmid for sequencing, you need to make sure it is clean. No protein junk, RNA, genomic DNA etc. Also Ideally the plasmid should be resuspended in water. Depending on protocol you need 0.5 microgram to 1 micrograme of DNA. Ask your postdoc, for help here. It is a little for an art to get good long sequence reads.
And no, you can not use the mutation primer to do the sequencing. The sequecing reaction will never read the mutation point if you do. (the mutant primer is sitting on the spot which you want to sequence. You need a primer that bind away from the mutantion point, but not far away. Maybe 100bp or so away. To be extra careful you may want to sequence the rest of your gene to ensure that there are no other mutations in the gene. in this situation the mutation primer can be used as an additional sequence read area.
A sequence read at the very best is 1200bp long. A less then perfect reaction, will have 500bp read lenght or less.
I hope this helps
For sequencing plasmids I usually just do a miniprep from an overninght 2 ml culture, use qiagen's miniprep kit (with the extra wash step), elute in 50 µl of 10 mM tris and use 1 µl in the sequencing reaction (Big Dye terminator). No problems so far.
So, as perneseblue stated, try some other competent cells (expensive) and browse this forum for increasing plasmid yield, some tricks are to elute in pre-warmed elution buffer, but this will not increase your yield 5-fold or so...