Hello. I recently cloned some plasmids for shRNA. I ordered PAGE-purified shRNA duplex oligo, and inserted it in lentivirus vector for shRNA(addgene cat.no. 8403, pLKO.1-puro) using AgeI and MluI enzyme restriction and subsequent ligation. after cloning, I checked whether there is very few number of colonies in vector-only plate(E.coli transformed using enzyme-cut vector) compared to cloned plate.
Before performing mini-prep, I checked whether the colonies in cloned plate actually contain desired, cloned plasmid. so, I performed colony PCR.
For the colony PCR, I designed forward and reverse primers that bind outside of shRNA. before cloning, the amplicon size is 270bp. after the shRNA is inserted, the size increase to 320bp.
graphical image of colony PCR strategy is attached below.
The problem begins in this colony PCR step. when I performed PCR using Taq polymerase based PCR mix, I always got two bands. when I used original vector as negative control, I got small size band only.
the result of colony PCR is also attached below. The leftmost band is the product of original vector(negative control).
So, what can be the possible reason of the two bands in the other lanes? (even though the rightmost band show very bright band at the upper side, there still exist dim lower band.)
At first, I thought un-restricted vector was mixed into cloned vector, since after enzyme restriction, I transformed E.coli with the restriction product, I got several(but lower number of )colonies. however, It statistically doesn't make sense that all the other colonies in cloned vector contains two different plasmids.
I have suffered from the issue since two weeks ago, and still I didn't found some clear answer. I hope anyone who know the answer or experienced similar issue give me any clue.
Thank You!
Hi In Kang ,
Try to perform a PCR without template to discard a contamination in your polymerase mix.
Have you tried setting up a gradient PCR with one colony and varying the annealing temperatures ? Do you get both the bands across the gradient ?
@Praveesh I didn't, since I always got single clear band at control plasmid as well as control plasmid transformed cell. I thought the possibility of non-specific band have the same size with the control band is low, so I thought those kind of gradient PCR is unnecessary. But now I think it is worth trying it.
I suggest to try primers in the vector. For instance 200 or 300 bp up en down-stream the insert.
José Tomás Cánovas-Márquez I tried PCR with no template or colony, but I got no band. so, the PCR mix is clear.
Praveesh Valissery I performed gradient PCR from annealing temperature 50`C to 65`C. I got no or very dim band at 60~65 lane, because the annealing temperature of my primer is 52`C. but, all the lane showed similar pattern(two bands).
Guillaume Van Eys That's good point, but since shRNA is only ~50bp long, if the total PCR product size is big, the small size difference is hard to distinguish. but I think it worth to be tried.
In Kang Can you redesign the reverse primer to include only the cloned insert ? The primers that you are using right now are probably picking up empty vector as well as your cloned sequence from the colony. Even if you think that is unlikely, the results suggest that might be the case with all the colonies. If your reverse primer is specific only to your insert, you should get just the single band for your clone.
Or pick a clone, isolate plasmid and send for sequencing. Since you are getting one of the positive bands, the insert should have been cloned in and the other band may be due to nonspecific priming.
I agree with Praveesh Valissery , If you try with a primer inside and other outside the insert you should obtain the positive result only in case of cloning.
In any case, double check that any of your oligos have a second homology region in the vector. I understand that you designed the oligos for U6 promoter region, which is full of A/T and CTS region, where you have a many G's repeated. The problem could be this oligos, that could hybridize in other regions of the plasmid.
Hi In Kang. My PhD project consisted in cloning libraries of shRNAs (112-bp in size) into recipient infectious clones to make RNA virus libraries. I can tell you I suffered with this issue of getting two bands from shRNA plasmids. We ordered a shRNA plasmid library (insert of same size but with different sequences) from the company and when I PCR amplified this library fragment the first I got were two bands (see attached gel image). I sequenced these two bands by Sanger and both (top band not matching expected size and bottom band matching their expected size) corresponded to shRNAs, not empty vector. Maybe try gel purifying both bands and send them for sequencing, running them in 2% agarose gel to separate them as much as possible and see what you get.
Hello In Kang,
In the past, I had to perform PCR on sequences that form stem-loop secondary structures, like shRNA do. Those sequences have an equilibrium between folded and open structure, and as such they can migrate as doublets. I thus agree that you could sequence both bands to confirm that they are two secondary structures of the same amplicon. Hope it helps.
- Badges
- Science topic
- Similar topics
- Vectorization