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Notebook: October 2011

Wednesday, October 12 2011

Vincent ran a PCR to amplify pSB3K1, which we plan on using as a backbone for the T7 + Lysis inserts. He used Expand High Fidelity Polymerase and its associated protocol for an expected band of 2774 bp. The gel to check it will be run on Thursday.

Thursday, October 13 2011

The gel for the K1 PCR amplification was run, and the results look good: a nice thick band right below the 3000 bp mark. Now we have to put the T7 + Lysis together with the backbone, and for that there will have to be some blunt-ends with phosphorylation before we can ligate the two pieces together. Stay tuned.

Sunday, October 16 2011

Vincent came in to work on the blunt-end cloning for the inserts (T7 promoters + Lysis) and the psB3K1 vector. The idea is to use the T4 DNA polymerase, which has a 3' to 5' exonuclease, to fill-in and chew away the ends till we have blunt ends. I added the protocol to the appropriate protocol section. After running through this protocol, the T7 inserts had an average concentration of 40 ng/uL while the backbone vector K1 had a concentration of 90 ng/uL (to within Nanodrop measurement error). Those concentrations are high enough to allow us to move on to the next step which is phosphorylating the T7 inserts (not the backbone!) for ligation.

The phosphorylation step is needed because the ligation (via ligase enzyme) requires that one of the 5' termini have a phosphate residue that will induce phosphodiester bond. If both the insert and the backbone have the phosphate residue, then the backbone may bind to itself, leaving the insert out -- which would defeat the purpose of ligation.

The specifics of the blunt-end protocol were as follows:

For the T7 + Lysis inserts,

• H20 - 11.25 uL
• NEBuffer2 (10X, found in enzyme ice box) - 3 uL (needs to be at a final concentration of 1X)
• dNTP (10 mM) - 0.3 (needs to be at a final concentration of 100 uM)
• template - 15 uL (for 1.5 ug of DNA, assuming the T7 + Lysis was at a concentration of 100 ng/uL)
• T4 Polymerase - 0.45 uL (it comes at a "concentration" of 3000 units/mL and we want 1.5 units)

I made a master mix of everything except the template DNA, which then went into separate PCR tubes. The total is a 30 uL reaction. The tubes were placed in the Bio-Rad machine were we setup a protocol (called BLUNTEND) that keeps the tubes at a constant 12 C for 15 minutes. If you want, you can add EDTA to a final concentration of 10 mM (as advised by New England Biolabs) and heat activate the samples at 75 C for 20 minutes. We decided that it was not necessary in our case.

For the K1 backbone, the recipe was slightly different due to the higher concentration of blunted DNA we wanted to achieve: