This page describes the formation of the "anchoring segment" which is subsequently ligated to the insert oligo to create the base unzipping construct.
Anchoring segment diagram
The goal is to obtain a double-stranded DNA (dsDNA) fragment of about 1 kilobase pair (kb) length, with a digoxigenin (dig) label on one blunt end, and an unlabeled sticky end on the other. The method we used was to do a PCR reaction with a dig-labeled primer, followed by a restriction digest of a single recognition site close to the non-labeled end. The short fragment was discarded while the large fragment was purified by gel extraction (and / or PEG precipitation).
Our specific protocol was evolved from existing PCR recipes involving the plasmid pRL574 from the Bob Landick lab. It's kind of funny how many single-molecule tethering experiments have used this plasmid, without any need for the underlying gene it encodes. We, too, do not require this plasmid exactly, but use it for convenience. If you're going to redesign, starting with a commercial plasmid that you can buy large amounts of purified plasmid prep would make sense.
There are many possible alternatives for preparing this piece. One would be to restriction digest a plasmid and ligate a dig-labeled hairpin or duplex onto one end. This would probably work very well, and allow for multiple-dig labels and / or a hairpin to increase the anchoring segment stability. We have not yet tried this as of 2007.
pRL574 purified plasmid DNA, concentration about 1.5 nanogram / microliter. This is about a 1:100 dilution of miniprep DNA. We obtained this plasmid from Bob Landick lab. The sequence of the plasmid is here.
A forward and reverse strand primer pair is required. A dig label (expensive) is required on the forward strand. (Or reverse if you're using a different reaction and restriction enzyme.) Steve recommends AlphaDNA as a supplier of dig- and biotin-modified oligos. They provide high quality oligos and technical and customer service is outstanding. Victor is extremely helpful.
Koch et al. 2002 primers
- pRL574-F853-dig: 5'-dig-GTAAAACGACGGCCAGTGAATTC
- pRL574-R2008: 5'-CACGTAAGGTTTCAGAGATATATGGG
A possibly better alternative
If you are gel extracting (not relying on shortness of short fragment after digestion), this primer pair may work better. We've tried it with success in a different protocol (not in this anchoring segment):
- pRL574-F834-dig: 5'-dig-TTTTCCCAGTCACGACGTTG
- pRL574-R2044: 5'-CTACCAGTGCGCTCAGACG
June 2008 Recommendation
Steve Koch 06:18, 15 June 2008 (UTC):I would try pRL574-F834-dig with pRL574-R2008
Standard PCR mixes and Taq polymerase.
For the Koch et al. 2002 paper, we used QIAquick PCR Cleanup and / or Enzymatic Reaction Cleanup kit. Probably any kind of cleanup will be fine. The main goal is for the subsequent restriction digestion to work well. The future gel extraction will get rid of primers if this step does not.
About 30 Units of BstXI from NEB and NEBuffer 3
I used a small-ish horizontal gel electrophoresis apparatus (from Bio-Rad, I think). I used 25 ml gels, 0.8% agarose in 1x TAE buffer (usually 0.8%, sometimes higher percentage). Typically ran at 90V for about 30 minutes. I always loaded ethidium into gel, about 0.4 microliters of Ethidium Bromide per 25 ml of gel. (I don't remember concentration of stock EtBr--I feel like it was 10 somethings per some volume.) Source of agarose I don't remember our supplier of agarose, but it was a relatively high purity--otherwise, after gel extraction there were too many contaminants.
We used the QIAquick Gel Extraction kit from Qiagen, spin column version.
Before scaling up reaction, you will want to test these steps. Below, I will put the scaled-up protocol.
Perform PCR and cleanup
Here is a link to an excel file for the recipe: Image:Koch Lab dig, bio PCR example.xls
Same as this program This takes a few hours. Set machine to hold at 4C when done.
We used QIAquick PCR cleanup (or enzymatic cleanup). The entire 500 ul of PCR reaction is loaded onto a single column, so as to obtain maximum concentration of DNA. The columns are not big enough for this amount (after dilution with binding buffer), but you can just load the 5 tubes sequentially onto the same column by repeating the binding step.
At this end of this step, after diagnostic gel, you'll have about 10 micrograms of DNA in about 45 micoliters or more. The QIAquick columns have a maximum binding capacity -- we're shooting to max it out, maximizing final concentration at expense of losing some DNA.
Restriction digest DNA
Mix recipe according to above, and incubate at 55 degrees C (note elevated temperature) for 2 to 4 hours (Note this is beyond the amount of cutting recommended by NEB...both in terms of amount of enzyme and duration of cutting). 1 hour would perhaps also be fine, even with less enzyme. Cleanup with QIAquick enzymatic reaction cleanup OR proceed to gel extraction.
Color and inverted B/W image of PCR product after restriction digest and QIAquick clenaup. "P13" is the reaction we are referring to in this protocol. Gel is probably 0.8% TAE agarose.
In order to completely remove the small fragment of the restriction digest, we used gel extraction. A gel similar to above was run, except 5 small lanes were taped together to fit the almost 50 microliters + loading buffer. The 25 ml (small, about 10 cm x 10 cm) gel was probably run at 90 V for about 30 minutes. Band is cut out with razor blade, and DNA extracted with QIAquick Gel Extraction kit.
- Elution buffer: You will use this anchoring segment at fairly high concentration in future ligation reactions. Therefore, choose a buffer that will not screw up the future ligation. Unfortunately, I cannot remember exactly what I used. Possibly 0.1x Tris pH 8.0.
Store in the 1.5 ml microfuge tube from gel extraction at -20C. Freeze / thaw cycles probably aren't a problem.