Hi, as the title suggests i'm having some trouble getting a sequencing result from a shRNA dsDNA oligo that i've ligated into the pLKO-puro plasmid. I have previously sequenced this plasmid both with inserts (ligated by other group and resulting plasmid gifted to me) and with no insert (to check the plasmid was correct to begin with when I bought it in fresh). Sequencing is by sending away to Eurofins and prep is following their instructions, usually they are very good so I don't think its their problem. I've sent away plasmid from two separate minipreps now and I still get nothing.
I get about 20 bases before ligation, then the (destroyed) restriction site, then exactly 19 bases of my shRNA sequence, then 2 random bases and the sequencing abruptly ends. In total including the restriction site overhands 21 bases of my oligo shows up. The read quality report shows that the read is really good at first, and then suddenly nothing - usually I get around 800-1000 base reads and the last 50 or so bases are slowly decreasing quality. Does anyone know what is happening? The read seems to stop just before the loop (XhoI) could this be a problem? I was given this plasmid with a different shRNA sequence but the same loop and that was minipreped then sequenced just fine. I guess it seems the sequencing enzyme is dropping off because the plasmid has been cut at the XhoI loop, I don't know why this would happen and i'll run a gel to check for this.
Any suggestions would be appreciated. Thanks
Here's a brief overview of my protocol:
1 - Individual ssDNA oligos are resuspended in nuclease-free water and then annealed by adding equal amounts (by mass) to an eppendorf in annealing buffer, then heated to 98'C on a hot block for 10 minutes and then allowed to cool slowly to RT.
2 - The plasmid is double digested with NEB AgeI-HF and EcoRI-HF following manufacturers instructions (except digestion time was extended to 1 hour from 15 minutes) and purified by gel extraction using the Zymo kit - the correct weight products were observed on the gel prior to extraction. The yield was quite low (lab book not at hand, around 10-20% recovery) but product was confirmed by again running on a gel.
3 - The dsDNA oligo and linear plasmid were ligated using Promega T4 ligase overnight at RT in a 1:6 (100ng plasmid:insert) ratio. I know I should be aiming for closer to 1:3 but I forgot to dilute my insert.
4 - 2uL of ligation product was transformed into Stellar competent cells (Clontech) following manufacturers instructions, a +ve transformation control and linear plasmid -ve control was also transformed in separate reactions. These resuspended in SOC up to 0.5mL and incubated at 37'C 200 RPM for 1 hour before 100uL being plated on LB-amp plates. The plates were incubated at 37'C overnight.
5 - NO colonies were observed on the linear plasmid plate and single colonies were picked from the ligation plate and grown in 5mL LB-amp overnight at 37'C with 200 RPM shaking.
6 - 0.5 mL of the media was used to make a glycerol stock, the rest was minipreped using the invitrogen hipure miniprep kit following manufacturers instructions.
7 - The DNA pellet was resuspended in nuclease-free water and quantified with the nanodrop. The yield was around 500ng/uL (which is what we usually get) and the 260/230 and 260/280 ratios were good, we usually get around 1.8-1.9 for 260/280 and I remember I got just below this, something like 1.78.
8 - 150ng was sent for sequencing (Eurofins) with 2 uL 10pmol/uL primer as manufacturers instructions up to 17uL - I have previously used this primer and the parent plasmid to check the plasmid was correct to begin with and had no trouble with sequencing.