Blunt end cloning PCR product - (May/23/2007 )
I am having problems in blunt end cloning a 92 bp PCR product. This PCR product contains two different blunt end restriction sites (PvuII and EcoRV). I purify the PCR product with PCR cleanup kit from Qiagen, digest it with the restriction enzymes and then gel purify it in a 1.5% agarose gel. Afterwards, I do a ligation with 10:1 insert to vector ratio and use Stratagene ligase O/N. When I analyze the plasmid from the transformants they usually come empty, eventough I used the Antartic Phosphatasefrom Qiagen to dephosphorylate it. Any ideas of what could be going on?
PD Would you guys recommend the Stratagene ligase over the NEB ligase?
Please tell us what the "it" in "... even though I used antarctic phosphatase Qiagen to desphophorylate it" refers to. You should be treating the vector, but not the insert. Just checking. You can try ligating the vector alone as a measure of the success of your cutting and dephosphorylation -- there should be few transformants. I'd recommend the NEB quick ligation ligase, for 2 hours at RT. Precipitate the DNA from the ligation mix, redissolve in TE, and then transform.
i would prefer NEB ligase over any other ligase. Especially quick ligase kit from NEB is very good.
Yes, by "it" I meant the vector . Thanks for the advice. Is transforming E. coli DH5alpha directly with the ligation mix not recommended?
We add the ligation mixture directly to DH5 alpha cells all the time.
as recommended by phage434, definately do a ligation control on your dephosphorylated vector. See if your vector is able to self ligate. Definately try the quick ligase or get your hands on some PEG 6000, 15% final concentration. PEG is key to the quick ligase formulation.
Now if that fails, is your PCR product being digested to produce the blunt ends? If so perhaps it is an idea to redesigning and purchase a new set of primers to produce sticky ends.
And finally, if the ends are blunt by PCR, perhaps TA cloning might be considered.
Maybe the 10 to 1 ratio is too big and the insert could tend to concatemerize if too concentrated, especially if they are blunt ended on both sides.
try a lower ratio like 5:1 , or 3:1.
I don't see any phnol:chloroform cleaning of your insert after purification from the 1.5% agarose gel, but I assume that you do it.
Somebody also gave me the PEG advice and it actually saved me a lot of time as well.
Do you clean your vector after dephosphorylation with phenol:chloroform.
Basically one of the essential key to a successful ligation is that the DNA should be free of any protein at least on the ends that are to be ligated, otherwise this can imped the ligation procedure.
like doing ligation straight after digestion is not advised, doing it straight after dephosphorylation may also not be good.
Get the cleanest DNA ever and try it again.