User:Anthony Salvagno/Notebook/Research/2009/08/24/New Unzipping Constructs

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Useful Pages

SapI Ligations
NotI Ligations

Intro

I am getting the ball rolling and I want to do it flawlessly. For the next few days I will be aligning the OT and working on ligations. The point of this is to ultimately unzip good DNA hopefully in 2 weeks time. I spent a good deal of today reading old notes trying to figure out exactly what I need.

The Process

  1. Cut pBS with NotI and SapI
  2. Ligate Adapter and NotI HP (digested) to NotpBS
    • I'm pretty sure I have this product ligated together as A+A NotI in Ant's Box #1
  3. Ligate Adapter and SapI top+bottom adapters to SappBS
    • I don't think I have this product, but I do have the molar amounts needed to do the exact reactions as shown above.
  4. Run gel of product and gel extract

For both reactions, I am pretty sure I can run the reactions exactly like I had in the past (see pages above).

Step 1: Digestion

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Run digestion at 37C for 2 hours. There is some discrepancy that I had to deal with. The amount of SapI is not the same as the amount of NotI. In each case I think the amounts I used are overkill, but better to be safe than sorry. For NotI the stock in 15 units/uL and for the SapI it's 2units/uL. As you can see above, I used 4 units of SapI and 15 units of NotI. Another question about digestions, do I need to use BSA? Nothing on the NEB website or the manuals provided with the REs mentioned anything about BSA and said that the enzymes are in solution with BSA which leads me to believe you don't need it.

Ok, I don't have enough time to do anything else today, but tomorrow I want to run a gel to make sure the digestion went smoothly. I also hope I didn't need to CIP treat my plasmid, and I don't even know if I have CIP. After digestion, I just froze my product. Do I need to inactivate my enzyme with 20 min of 65C? The manuals of the REs say to do that. I usually do a P/C EtOH precipitation, but I don't want to make EtOH stocks (70% and 100%).

Helpful hint: Always do digestions, ligations, annealings, anything that uses the KL thermocycler in small reaction tubes because it won't fit the big ones.

Where to find things I need for the future

"pBS Sap" and "pBS Not" are two tubes in my newly minted Box #3. They have the non-cleaned digestion I did today. A tube labeled "anchor 4/21" has already been cut with BstXI. Tube "A+A Not" contains ligated anchor plus NotI Hairpin adapter, it might be predigested with NotI, but a gel of the final products against this segment would reveal the truth. "T+B" with some molarity in green is the molarity that I need for the required reactions. Also pBlueScript is also in Box#3.