blunt-cloning - all are vector self-ligation products? (Jul/11/2003 )
besides the previous answers, CIP treatment and proper purification of both, vector and insert, take care on the ligase buffer...after several defrozing steps, ATP could be depeleted. I improved results adding ATP 10 mM to ligase reactions. If the buffer is not so old, aliquote it in small amounts, just to use once or twice.
To stimulate the blunt-end reaction, 150-200mM NaCl and 5% PEG can be added.
As above, but you can either dephosphorylate your vector with SAP, or phosphorylate with TAP and T4 polynucleotide kinase.
Hi...Karen. Have you solved your problem?
There are some factors that influence result of Blunt-end ligation, including de-phosphorilation of vector, vector purification after de-phosphorilation etc. As usual ligation, you also should pay attention on ration of insert and vector concentration. In my experience, BAP (Bacterial Alkaline Phosphatase) also work well for de-phosphorilation of vector.
[QUOTE].after several defrozing steps, ATP could be depeleted. I improved results adding ATP 10 mM to ligase reactions. If the buffer is not so old, aliquote it in small amounts, just to use once or twice
I am doing blunt-end ligation too and also have the trouble to get the clony. You suggestion is very good and I will try it. I am eagerly to know that why the ATP could be dpeleted after using several times. Could you tell me the answer?
Generally ligases come with buffers for cohesive ends and also for blunt ends. So check out the buffer you use. Also, 10 times higher liagse quantity is advised for blunt end ligation.
The vecotr insert ration should be atleasr 1:3
As blunt-end ligations are quite inefficient, dephosphorylation will make it even more inefficient.
Personnally, I won't de-phos at all. Yes you'll get squillions of ligation positives but you can screen 50 - 100 very easily using single-colony gels (or blue/white selection even better of course if you have it). A single colony gel (if unfamiliar) is simply a blob of +ve colony lysed in SDS buffer (with a dye), spun and ran on a large agarose gel. An insert will cause the supercoiled plasmid to run slower than plasmid w/o insert. I've used this technique to detect a 50bp insert in a 6kb plasmid.
Way I did it (successfully):
- purify insert + vector with prefered method
- IF cutting with the same REs, mix plasmid + vector together and digest
- purify with prefered method
- add salt, dNTPs and Taq, incubate for a couple of hours
- clean up again
- add ligase + buffer etc and incubate o/n
- don't forget to pray between steps :-P
hi... do you have the protocol using Tag... I mean, dNTPs concentration, enzime units, DNA concentration, Temp, etc. I read it's a good alternative to klenow or S1 nuclease. Thanks
I would be interested in the single colony gel method. Could you plz send me a protocol for how to do it?
I am doing a very tricky blunt-end cloning trying to get a 3 kb fragment into a 12 kb vector.
Maybe the dephosphorylation makes it too difficult as you say.
I do blunt cloning directly from PCR products and don't really have problems. MMM...If CIP is not working very well maybe try Antartic Phosphatase by New England Biolabs.