Cloning 1.5kB gene into lentivirus--I'm stumped - (Jun/06/2008 )
Hello all,
I am trying to clone a 1.5kB gene into a lentiviral vector (has ampR gene) that we use routinely in the lab. I've troubleshooted through a few problems over the last two months, but now I'm stumped and I need help! I'm relatively new at this, but even the molecular bio guy in our lab who does this all the time doesn't know what to do. Here's what I have done so far:
PCR gene with primers adding BAMHI restriction sites to each end
obtained PCR product of correct size
cloned this into TOPO successfully (I got colonies) using TOP10 chemically competent cells, selected 4 colonies
digested vector and plasmid with BamHI overnight at 37C
dephosphorylated vector with CIP for 1h at 37C, cleaned with PCR clean kit (Qiagen)
ran plasmid digest on gel, obtained fragment of correct size in all but one digest
purified gel fragments using Promega wizard extraction kit
set up ligation reaction with 1:0 and 2:1 ratios of vector:insert (approximately 150ng total DNA in a 10uL ligation reaction volume)
ligated overnight at 16C
used 5uL to transform ~30uL TOP10 competent cells
incubate in 1mL SOC for 45min, then plate 250uL on LB-Amp plates
the next day I got 5-10 colonies on each 2:1 plate and very few or no colonies on the 1:0 plates
From those plates, I selected 12 colonies (4 from each 2:1 plate), grew those up in LB-Amp for 6h and digested the DNA overnight at 37C with BamHI to check for the plasmid of the correct size. Of the 12, I only got one that had an insert of the correct size, and sent it for sequencing, hoping it would be in the proper orientation since I was using a non-directional strategy.
Now here's the kicker--the sequencing showed that the insert appeared to be of the correct orientation, however the gene was truncated at about 430 bp, and the rest of the DNA was junk! It wasn't from the vector, and there is no Bam restriction site where it was truncated...
Could this be from plasmid recombination or what? And based on the fact that I only got 1/12 colonies with any part of my insert, I think something else must be wrong...
Any suggestions would be greatly appreciated!! Thank you in advance for your help.
The one thing in your protocol that I don't like is the overnight BamHI digestion. BamHI has star activity and during a long incubation the water condenses on the tube leaving the actual digest more and more concentrated. What tips off this idea is you mentioning that most of your inserts were not the correct size. When you analyze the "junk DNA" by BLAST analysis what does it come up with? What about if you reverse the sequence and then BLAST? Unless you were specifically looking for it, it would be difficult to identify a fragment of the vector (a star digestion product) ligated in the reverse orientation. I would suggest starting over with a much shorter digestion. BamHI is a good enzyme that shouldn't need that long to get an effective digestion. Gel purify your vector before Cip treating. Cip adds more glycerol to the digest which can activate star activity. Gel purify, then Cip and clean with a pcr purification.
Your ligation ratios are pretty bad as well. You wrote you did a vector:insert of 2:1 but I am hoping this is a typo!!! You want a vector:insert ratio of minimally 1:3. I usually set up a 1:4 and 1:5 as well. This may explain why you had so few colonies and the digest potentially explains why your inserts are wrong. Hope this helps.
Your ligation ratios are pretty bad as well. You wrote you did a vector:insert of 2:1 but I am hoping this is a typo!!! You want a vector:insert ratio of minimally 1:3. I usually set up a 1:4 and 1:5 as well. This may explain why you had so few colonies and the digest potentially explains why your inserts are wrong. Hope this helps.
Rkay, thank you very much for your helpful suggestions! I think I will start by making fresh vector as you suggested by gel purifying before the CIP treatment, then pcr purify, and repeat the ligation with a higher proportion of insert to vector than the 2:1 I used before (I'm sorry, the "vector:insert" was a typo in my original post! I've been thinking of it in terms of insert:vector--you need more molecules of insert than vector for optimal ligation, is that correct?). Is "vector:insert" the standard terminology for it? So I will try 1:3 and 1:5 probably. Like I said, I am new to this, so thank you for bearing with me
.As for the "junk DNA", I did check by BLAST and it doesn't match anything forward or reverse, but I will double-check on that, because it does sound like a good possibility that I had Bam star activity in my vector digest.
In the meantime, I will also go back to my TOPO/insert plasmid and digest for a shorter period of time. You make a very good point about the digestion probably being too long for Bam, however even after overnight digestion of my TOPO/insert plasmid with Bam, 3 out of 4 digests at that point had an insert of the correct size so I would suppose that there was minimal star activity in those. Both the insert plasmid and the vector were digested in 100uL volume with 20U of Bam, so perhaps as you suggested it was the addition of CIP that could have affected the vector. Would it be helpful to reduce the amount of Bam used in addition to shortening the incubation time of the digest?
Thank you again for your excellent suggestions--I will update with my progress next week!