strange transformation products in subclone - (Dec/01/2007 )
I worked on subclone with expression vector. After ligation and transformation, I usually get some colonies (vary from 4-100). But as I digested the plasmid from growing colonies on the plate with restrict enzymes (using the same pair of the enzyme as on insert taget gene and vector before ligation), I found many clones with incorrect size as I expected and the small size band is brighter than the large size one. I want to know what is the reason to cause this. Ratio of ligation? transformation time? plasmid extract problem?
I can find 1 or 2 clone positive sometimes.
I have another question. If I digested the PCR product and vector with restrict enzyme, then do not run the gel for purification. extract them by Qiagen PCR purification kit instead. Do you think this is the problem?
Normally the insert should be dimmer than the vector. If it is not, perhaps you have cloned a plasmid with multiple copies of your insert. Even with directional cloning, three inserts can form a concatamer and insert. Lower the amount of insert in your ligation. The insert and vector should be equimolar and the dna concentration should be relatively low, around 10-20 ng/ul at most to favor single insertions and recircularization.
Thanks for your analyse and suggestion. Do you mean that I should add 150ng insert and 120ng vector (suppose it is 1:1 in ratio of molar) in 40uL ligation system (270ng/40uL=7ng/uL)?
just to confirm things, is the insert use for the ligation the correct lenght? ... just checking.
As for you last question, it is okay to sequence PCR products without gel purification as long as there is only a single clean PCR product in that reaction, with no secondary bands or heavy smearing. If the PCR product is not clean, then a gel purification to excise the desire band is required.
A 20ul ligation reaction with 20ng of vector and a 1:1 ratio of insert should be fine.