Koch Lab:Protocols/Unzipping constructs/17mer/Anchoring segment

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This page describes the formation of the "anchoring segment" which is subsequently ligated to the insert oligo to create the base unzipping construct.

Overview

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.

Alternate methods

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.

Materials

Plasmid

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.

Primers

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

PCR Materials

Standard PCR mixes and Taq polymerase.

PCR Cleanup

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.

Restriction digest

About 30 Units of BstXI from NEB and NEBuffer 3

Gel extraction

We used the QIAquick Gel Extraction kit from Qiagen.

Procedure

Before scaling up reaction, you will want to test these steps. Below, I will put the scaled-up protocol.

Perform PCR and cleanup

Recipe

Here is a link to an excel file for the recipe: File:Koch Lab dig, bio PCR example.xls

Thermocycler program

Same as this program

Restriction digest DNA

Gel extract