Ligation problem - ligation (Jun/17/2010 )
In the gel from the right after Marker (WHITE Pict)
1. the vector linearized with BglII(pAB NT) NOt gel purified just pcr cleaned
2. Insert for NT
3. Gel purified Ct (BglII XbaI cut pAB)
4. pcr cleaned not gelpurified ( BglII XbaI cut
5. CT Insert
your gel seems to be fine!
i must confess i do not really get what you are trying to do ...but sometimes it is hard to explain not being in a face-to-face situation.
i would recommend setting up a ligation reaction with only the two inserts and without vector ...and load those two reactions on an agarose gel to check if both inserts are digested properly (you can post that gel pic here if you want to).
If you vector backbone is really dephosphorylated 100% (since you do not get colonies) it is essential that your insert is correctly digested ...so you have to check this first.
Further it would be interested who you introduced the formermentioned mutations in your vetor backbone? By PCR mutagenesis? ...maybe you destroyed some essential part in the origin of replication of your vector and therefore you are unable to generate colonies?
In the Gel The 2 bands in the lower marker range around 3.8 to4kb (it ispromega 1kb ladder )they are the two inserts one is from Nterminal another is cterminal almost the same size
All I am trying to do is swap the toxic gene with other essentail portion of the vector which is created as destination vector for GAte way cloning for mammalian expression into the baculovirus vector so that we can transfect the naked dna into baculovirus system once they spit the naked dna with viral component that can be injected into mammalian cells with out introduction of any transfection reagents like PEI or other liposome regents which we are experiencing much difficulties (Have you heard of BAC MAM system thats what we are trying to do)
When we cut the insert we left the ORI part so it doesnt have any.
For cloning my insert into BAC system we did nothave sites compatible to the Baculovirus vector. In the MCS of pAB Vector we chose XBaI and BglII to clone our insert. Since our insert doesnt have this we had to introduce by mutation ( I did point mutation although it should be st fwd procedure it was difficult due its repetitve seq pattern of our pXLG vector)
why you did not amplify your insert using PCR and add the restriction sites on the PCR primers? ...this would be more straightforward?
I did PCR to intoduce my mutation having restriction sites on my PCR primers
that messed somepart although gave my mutation my boss says it is the original design of the cons has somemany repeats like ATTTATATATATTTAAATTA atleast 5 or 6 repeats which made it so difficult for the mutation to get it done
idid introduce my mutation thr pcr primers
messed up but gave my mutations i think repeats in cons is the problem
okay ...try to check the ligation on an agarose gel ...and then we will follow up!
if i run it on the gel i should see the 9.6+3.8kb total of 13kb size pdt? is that right
if your insert is 3.8 kb than you should see a band at 7.6, 11.4 kb and so on ...the same for the vector and ...a kind of ladder
you can do this as well for the insert and the vector seperately ...then it should be more clear.
The theory is that vector and insert are ligating with itself if they are proper digested and you get dimer/trimer and so ...if you only get the band for the dimer (size=2xinsert size) than you can assume that only one end of your insert is digested.