Help with Cloning into LARGE vector - (Jan/05/2007 )
Hello, I am brand new to this forum so thank-you in advance for your help.
I have been trying for quite a while to clone into into a very large (12 Kb) vector (that contains two cassettes with two different flourescent genes with their own promoters - plus it's a T-dna for use with agro infiltration). The inserts I am using are 2 and 3 Kb. I have tried many different approaches starting with PCR of the insterts and restriction digest (Xba1/Sac1) of the PCR product. Then gel extraction using a Quaigen kit.
The vector I maxiprepped using a Quiagen kit and eluted with TE. Then digest with Xba1/Sac1 which drops out a 700 bp band. I then gel extracted the linearized vector to use in the ligation.
I have tried many different ratios of vector to insert, different batches of buffer and ligase etc. and incubation overnight at 16 degrees celcius. Everytime I do the ligation my negative control plates are clean, and my ligation plates (on the appropriate antibiotic) have about 20-70 colonies. PCR screens always show false positives (proven later using diagnositic restriction digests) or no positives, but the colonies are not empty vector either.
I have tried cutting out the 700 bp band and re-ligating it into the vector which works fine.
Recently I cloned one of the genes into bluescript (and had it sequenced) and then cut it out of bluescript and did a ligation with the large vector. I screened all 40 colonies on the ligation plate by colony PCR and none came up positive. I miniprepped the DNA from three colonies grown overnight just to see what they are and once again restriction digests show they are not empty vector, nor do they contain my gene.
Any suggestions on some other approaches I could try?
1- gel purify your cut vector twice. This should remove any contaminants.
2- gel purify your PCR products twice. From the description it sounds like your vector is picking up an insert from somewhere. I am guessing the contaminating insert is coming as a hitch hiker from the PCR insert.
Any idea what these false positives are? Are they the vector parent?
Hmm...
You might consider growing your cells at a lower temperature, around 30 - 32 Celsius. This could also be cause of recombination, ie the insert is not stable within the cell. What strain of e coli are you using? Is it a proper cloning strain rather then an expressioni strain.
2- gel purify your PCR products twice. From the description it sounds like your vector is picking up an insert from somewhere. I am guessing the contaminating insert is coming as a hitch hiker from the PCR insert.
Any idea what these false positives are? Are they the vector parent?
Hmm...
You might consider growing your cells at a lower temperature, around 30 - 32 Celsius. This could also be cause of recombination, ie the insert is not stable within the cell. What strain of e coli are you using? Is it a proper cloning strain rather then an expressioni strain.
That's a good idea with the double gel purifications - I'll try that.
I can't figure out what the false positives are! The vector was a gift from another lab that was put together from a couple of places - I have a rough map but no sequence, and no primer sites for sequencing.
With the subcloned insert, I cut the band out of gel and purified and still had the same problem so could a contaminating insert still be hitching a ride with that? Wouldn't I be able to cut out whatever is hitch hiking in with the enzymes sites I'm cloning into?
I'm transforming into TOP10 cells (originally from Invitrogen but home made competents).
thanks
i advise dont use maxipreped vector for cloning. do very clean miniprep instead. i had much trouble with maxipreps. another thing is can't you find some restriction enzyme site inside the insert (not yours the one that you dont want) and after you ligate, treat the ligation mix with that enzyme. be careful that it doesnt cut anywhere else inside the vector or your DNA. like that all the clones that you dont want will be linear and wont get into e-coli. hope that helps.
ah and another thing i forgot to mention. if you see PCR positive result and no insert on the digest, that doesnt necessarily mean that they are false positive. sometimes PCR modifies the enzyme sites and you cant cut with them anymore. try different flanking enzymes. if there arent any, cut with only one enzyme and see if your vector is increased by the size of the insert.
Well, if the PCR insert was run as a concentrated enough band, it can pull down other DNA fragments in significant quantities during the gel purification. That may be the cause.
Though this doesn't really answer the observation made when the PCR insert was first cloned into the pbs and later cut out to be ligated with the vector....
This is troubling. The stain seems okay. Still in your next experiment, would it be possible to grow your cells at 30 Celsius. It will take longer but humour me.
Cloning into large vectors as well these days, inserting severall kb into vectors of over 10 kb with 2 viral LTR's (making the DNA unstable).
If I were you, I'd clone your PCR product's into TOPO/bluescript/PUC... Sequence all of it. Then cut out your fragment and gel purify without UV-exposure. (run a small part of your digest on the gel, and the rest severall lanes further, so that you only have the small part exposed to UV, excise the other band). RUn the extracted DNA on gel to make sure all is all right and check your A260/280 ratio.
Have your vector treated the same way (meaning gel purification). As it is a large vector, make a large gel and run it for several hours on low voltage to get clean separation. I know that for instance qiagen gel extraction is only supposed to work up to 10 kb, but most likely larger fragments will also work. I've used Invitrogens SNAP gel purification kit up to 12 kb with huge succes. Check on gel, determine concentration. Perform ligation and proceed with transformation, perform all growing steps @ 30°C and shake at 180 rpm after heat shock for 90 minutes (instead of 37°C and 225-300 rpm for one hour as you most likely do?). Incubate at 30°C after plating (you'll get smaller colonies, so grow them longer).
Has worked many times for me, succes ratio's of 15/20 and 1-2 colony's on negative control plate.
Good luck!
IF possible, try to use a different strain of bacteria than the one u ur using. U could try strains which prevent recombination like SURE cells or Stbl3.
Good luck !!!
Thanks everyone - will try all suggestions and let you know how it goes.
Lori
Update!
Ok I finally got one of my genes into the large vector I've been having trouble with.
I double gel extracted the vector - and had subcloned my gene into bluescript and then cut it out of there. I did the ligation at 16 degrees for 22 hours and then 4 degrees for 2 days.
It turned out that a visiting student had made a new 50X stock of TAE for the DNA gels and hadn't pH'd it after adding the EDTA. The pH was 4! So I think that was actually damaging the DNA I was gel extracting and preventing it from ligating.
Thanks for all your help guys!
Lori, finally making progress in Calgary