3 replies to this topic

[ana71]

hi,
i am new to cloning and having a bit of trouble. hope you can help
i am trying to clone different inserts into pxJ41 for splicing assay (ie minigene)
these are the details:
clean PCR products of correct size digested with EcoRI
vector cut with MfeI and dephosphorilated with anctartic phosphatase, gives correct band on gel
ligation performed ON at 4°C (Promega T4)
ligation products transformed into JM109 v:i ratio 1:3

i get no colonies in my negative control (ie no insert) and plenty of colonies in my test plates. however when i screen those, they are all empty!
i have used the same protocol for different inserts and it does work ok for some of them, but there are 2 inserts that i would not go in!

what could be going on?
many thanks!!!

[swanny]

ana71, on Feb 3 2009, 12:54 AM, said:

hi,
i am new to cloning and having a bit of trouble. hope you can help
i am trying to clone different inserts into pxJ41 for splicing assay (ie minigene)
these are the details:
clean PCR products of correct size digested with EcoRI
vector cut with MfeI and dephosphorilated with anctartic phosphatase, gives correct band on gel
ligation performed ON at 4°C (Promega T4)
ligation products transformed into JM109 v:i ratio 1:3

i get no colonies in my negative control (ie no insert) and plenty of colonies in my test plates. however when i screen those, they are all empty!
i have used the same protocol for different inserts and it does work ok for some of them, but there are 2 inserts that i would not go in!

what could be going on?
many thanks!!!

How do you screen? Colony PCR is notorious for giving false results, unless one of  the primers is from the vector and the other is from the gene. Restriction digests work well. If you can find a site that is in the insert, but not in the vector you have a good assay. Cut with that enzyme and run out: if there is an insert, the plasmid is linearised, if not it remains supercoiled.

Do you test to make sure the insert is cut properly? Add ligase to some of your "digested, cleaned up" insert for ~20 minutes at RT and run out. If you don't get a ladder/smear, your enzyme is dead / sick.

[TanyHark]

ana71, on Feb 2 2009, 06:54 AM, said:

hi,
i am new to cloning and having a bit of trouble. hope you can help
i am trying to clone different inserts into pxJ41 for splicing assay (ie minigene)
these are the details:
clean PCR products of correct size digested with EcoRI
vector cut with MfeI and dephosphorilated with anctartic phosphatase, gives correct band on gel
ligation performed ON at 4°C (Promega T4)
ligation products transformed into JM109 v:i ratio 1:3

i get no colonies in my negative control (ie no insert) and plenty of colonies in my test plates. however when i screen those, they are all empty!
i have used the same protocol for different inserts and it does work ok for some of them, but there are 2 inserts that i would not go in!

what could be going on?
many thanks!!!

I think I know what is your precise problem!!



Do O/N incubation at 14'C.

[scolix]

How much DNA do you use for the ligation? Typicall, we use 20ngs of vector.

I suggest trying a little higher amounts of DNA like 50 ngs and also try to ligate it at RT for 1 hr atleast.

Good Luck !!!