User:Anthony Salvagno/Notebook/Research/2011/05/11/Creating the New Unzipping Construct
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Unzipping Construct
The unzipping construct consists of 3 pieces. It has a dig end-labeled anchor segment (the above description), there is unzipping DNA which is the segment of DNA we wish to probe, and there is an adapter segment that joins the two together.
Anchor PCR
I designed a custom plasmid from DNA 2.0 to have a specific sequence that could be useful in it at some point. It also has a BstXI recognition site in it for ligation in a later step. The plasmid sequence and info can be found here: Plasmid Sequence.
The first step is to create a PCR fragment from this plasmid. The primers I created and use can be found here: Primers.
Note: Use the forward primer and the new reverse primer. If you use the old reverse primer there is a high likelihood of getting 2 products for your PCR reaction.
My PCR reaction setup can be found here: PCR Reaction.
The reverse primer has a digoxygenin molecule attached to it which is used to tether our DNA constructs to a glass surface coated with anti-dig molecules.
Finally a simple digestion reaction prepared with the PCR product and BstXI will cleave the BstXI site located near the forward primer producing an overhang.
Adapter Duplex
The adapter piece contains a biotin labeled nucleotide and a nick. The adapter is actually made by annealing two oligonucleotides. They form in such a way that one side produces a BstXI overhang (complementary to the overhang created in the anchor construct digestion) and the other side contains a SapI/EarI overhang complementary to an overhang we will produce in a bit.
Just mix the two oligos in annealing buffer in close to boiling water (or a thermocycler) down to room temperature or 4C and voila! Adapter duplex is ready.
Unzipping DNA
This is the DNA that we will probe. We start with pBR322 plasmid DNA which is readily purchasable from NEB. From here you can do one of two things:
EarI Digest Method
Prepare a digestion reaction with the plasmid DNA and EarI. Upon completion run a gel to separate the two product strands since there are two cut sites for EarI in pBR322. We gel extract the larger of the two bands and then purify it.
SapI Digest Method
Prepare a digestion with pBR322 and SapI. This will produce 1 linear band so no gel analysis is needed except to check for completeness of digestion.
Ligating Products
Depending on your unzipping DNA prep you will need to do a ligation reaction to ligate each separate piece to create one linear DNA molecule that is ready for tethering and unzipping.
EarI Prep Ligation
If you digested with EarI and gel extracted then you can set up a 3-piece ligation. Put in equimolar amounts of unzipping DNA and anchor DNA. Start with a small amount of adapter duplex (maybe 1/6 the molar quantity as the other two) and start the reaction. Every 20-30 min add the same amount of adapter that you started the reaction with. Do this until you match and exceed the molar amount of anchor/unzipping DNA.
Here is a sample protocol: Ligation Protocol
After the reaction, you will run a gel and extract the band that is the total length of the construct. For instance the anchor piece is ~4kb and the EarI fragment is ~2.5kb. So your product should be around ~6.5kb.
SapI Prep Ligation
If you digested with SapI then you will do two separate ligation reactions. The first will be to ligate your SapI digested pBR322 to the adapter duplex (or ligate the adapter to the anchor). Place an excess of the adapter to prevent the plasmid from self annealing and from re-circularizing.
After reaction cleanup prepare another ligation with your reaction product and the anchor DNA. After this reaction is complete you will run a gel and extract the band that is the total length of the product.
Notes
Digestion enzymes, and DNA Ligase purchased from NEB. Taq purchased from Invitrogen. Reaction cleanup performed using Qiagen reaction cleanup kits. Please direct questions to me (see user page).
More can be found here: Unzipping Construct protocol
