clonning problem - (Dec/21/2004 )

hello guys.
i am having a cloning problem. i am currently trying to clone 1.2kb insert and 1.7kb inserts into 3.4 kb vector. I pcred those inserts and digested
with the enzymes that i was supposed to. i also digested the vector overnight one by one. i ligated them in 4 different ways. 1:1 using 25ng, 3:1 using 50ng, 1:3 using 50ng, and maxed out inserts. Then i transformed into M15 and i saw good amount of colonies(about 10/plate) where control plate(self ligation control) was good. I innoculated those and did the DNA extraction and i see a degraded bands. I thought it was the TAE buffer or the apparatus, so i claned everything and it still yields the same result. Any suggestions??

hi,

How long time did you use to digest your insert and vector, overnight? After that , did you purify them? Were they at the right position? Did you check the RE site of your vector and insert? I am not sure the exact reason, but it should be one of the above from my apart. Good luck for your research!

Daniel

thanks for your input Daniel.
However, i did everything that i could've done. I digested the inserts
for about 3 hours and ran on the gel and got a right size band. On the vector, i digested over night and ran on the gel and got the right size. some people say it might be that the insert is somewhat big, but i see other people clonning 2kb inserts without any problem. I NEED HELP!!!