linearisation of plasmid - pBS never gets cut :-( (Oct/07/2005 )
QUOTE (chguser @ Oct 17 2005, 12:12 PM)
insert is cDNA thats ligated with ecor1 adaptors,which is further digested with ecor1 that would give ecor1 and xho cohesive ends.
Are you sure that your adapters ligation worked correctly ? 'cause it might be the explanation if you have only partial ligation !
How do you generate XhoI extremities with EcoRI adapters ? Is there a XhoI internal site into your adapters ? Please gave us more clues on the design of the experiment that we can help better
pesji
-pesji-
QUOTE (chguser @ Oct 17 2005, 12:15 PM)
QUOTE (pesji @ Oct 10 2005, 06:27 PM)
Never forget that if you have still a little bit of supercoïled vector (bad cutting of one enzyme) you will surely trap some into your vector purification specially if you overload your preparative gel. I had this problem several times
Just to remind you
pesji
we cant have the supercoiled dna as we do a pcr purification after the xho digestion and a gel elution after the ecor1 digestion.so i dont think that should be a problem.
Oh yes you can you would be surprised how much you can trap with a gel elution even if the bands are quite different in size ! If your digestion is not complete then you can be sure that some of the supercoiled will be around !
-pesji-
QUOTE (pesji @ Oct 17 2005, 04:08 PM)
QUOTE (chguser @ Oct 17 2005, 12:15 PM)
QUOTE (pesji @ Oct 10 2005, 06:27 PM)
Never forget that if you have still a little bit of supercoïled vector (bad cutting of one enzyme) you will surely trap some into your vector purification specially if you overload your preparative gel. I had this problem several times
Just to remind you
pesji
we cant have the supercoiled dna as we do a pcr purification after the xho digestion and a gel elution after the ecor1 digestion.so i dont think that should be a problem.
Oh yes you can you would be surprised how much you can trap with a gel elution even if the bands are quite different in size ! If your digestion is not complete then you can be sure that some of the supercoiled will be around !
so what do you think is the reason that its not getting completely digested when we add the 100 percent activity buffer?the incubation time is also pretty decent, 6 hours for xho and overnight ecor1
-chguser-
I agree that everything looks perfect but you know after 25 yeras in a lab I've seen so many unexplained things that this one doesn't surprise me that much. Possible causes might be purity of DNA, presence of inhibitors, bad stock of enzyme etc...
You didn't reply for the adaptor story how you did the overall thing ?
pesji
-pesji-
QUOTE (pesji @ Oct 17 2005, 03:47 PM)
QUOTE (chguser @ Oct 17 2005, 12:12 PM)
insert is cDNA thats ligated with ecor1 adaptors,which is further digested with ecor1 that would give ecor1 and xho cohesive ends.
Are you sure that your adapters ligation worked correctly ? 'cause it might be the explanation if you have only partial ligation !
How do you generate XhoI extremities with EcoRI adapters ? Is there a XhoI internal site into your adapters ? Please gave us more clues on the design of the experiment that we can help better
pesji
im sorry,i had actually given a wrong information.to the cDNA we add the ecor1 adaptors and then digest it with xho1(there is an internal site for xho at one end of cdna) which would give ecor1 and xho ends and as you said even i was thinking of ecor1 adaptors really getting ligated to cDNA, so gonna do a PCR as well to check if the adaptors are ligating...would get back to everyone after the experiment
-chguser-