ligation into pcDNA3.1(-) - (Mar/11/2006 )
Hi everyone
I have a big problem cloning my insert and I think i'm going to get insane!
I have a insert (2.3Kb) into a PCR2.1 plasmid..I digest it with KpnI and XhoI (neb) this way its in the right direction...in the Neb buffer 1...
I do the same with my pcDNA3.1(5.4kb) vector (I verified it matches....)
I load it on a 0.8% agarose gel with a 1kb ladder then I cut my band of interest and extract the DNA with the QI extraction gene kit!
I verified with an agarose gene I have the right insert and my linearised vector...
I do a ligation with the T4 ligase with a vector:insert 3:1 ratio at 14 degre overnight with my insert+vector and as a control: my insert cut with the two enzymes with no other treatment (so it can re-ligate on itself)
I transform inv-alfa cells from invitrogen with 2uL of my vector re-ligated on itself, my vector+insert ligation, and even the pUC19 vector that invitogen gives(1uL) on ampicilin treated LB-Agar plates.
I put everything at 37 DEGREES OVERNIGHT ...
i DID ALL THESE many times and my control transforemd cells grows(vectopcDNA3.1 and pUC19) but NEVER MY INSERT-VECTOR transformed cells.
I changed everything my competen cells, my ligase, even the vector but they obviously works!!!!
so THE QUESTION IS why it still DOES'NT WORK???
I don't see what I can do wrong...
PLEaSE GIVE ME some IDEA...
i'M ON A 5 MONTH PROJECT WITH AN oral defense at the end to valid my master degree and i already spent 1month on that part and i can't go forward without this
I really need help
What gene is it that you are cloning? Maybe it is toxic to the cells that are producing it. Also did you check that you had successfully ligated by running a gel and looking for different size bands? Maybe ther is no ligation product!
I have the same problem with the ligation of a 3kb insert to pcDNA3.1(+) vector. I digested Insert by SalI and XbaI; vector by XhoI and XbaI. Is it possible that it's difficult to get pure vector fragment digested completely? Cuz these two enzyme sites are too near. How about checking the remanent vector fragment by selfligation without other fragments and doing transformation? If it's really not pure, how can I get the pure vector fragment. I got it from double enzyme digestion and then gel elution. But the size of completely digested vector and uncompletely digested vector is almost the same. It's really annoying. Can someone give me some idea? I really need help very much since I couldn't start doing other cloning unless I get this ligation products
Thanks in advance
\I don't think the gene is involved , but for info its "alcam".I'm pretty sure its lethal because i did mini preps of it in PCR2.1 in the same competent cells and I had a lot of colonies
i was thinking maybe i should change competent cells ...I don't for some reason maybe they just can't 'absorb" my vector with my insert???(a queation of size?)
Is that possible ?