User:Morgan L. Paull/Notebook/C.Dog V1.0 - T7 Promoter/2011/06/21

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] C.Dog T7 Promoter Project
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Nanodroped GFP and RFP backbone plasmids

 * Concentrations a little low, in the 15 to 60 μL range.
 * Next round of minipreps I will do only one miniprep tube per culture tube, to increase concentration

GFP

 * 50 μL G1.1 miniprep (~1500 ng)
 * 1.5 μL BsaI enzyme
 * 6  μL NEB buffer #4
 * 2.5 μL DH2O

RFP

 * 50 μL G1.1 miniprep (~2500 ng)
 * 1.5 μL BsaI enzyme
 * 6  μL NEB buffer #4
 * 2.5 μL DH2O

Began incubating each in 37°C water bath at 11:45 am, removed at 2:45pm, ran on .7% agarose gel, cut out the lowest band (digested backbone). Performed gel purification protocol, skipping isopropanol step at Colin's advice.

Oligos
Received in mail:
 * 1361 - Makoff1_FW_WT
 * 1362 - T7-promoter-FW
 * 1363 - T7-promoter-RV

Centrifuged tubes at 15,000 rpm for 10 mins, resuspended with (nmol*10) μL of DH2O:
 * 1361 at 26.5 nmol --> 265 μL DH2O
 * 1362 at 26.8 nmol --> 268 μL DH2O
 * 1363 at 33.7 nmol --> 337 μL DH2O

In PCR Tubes

 * 1 μL T4 Polynucleotide Kinase (PNK)
 * 2 μL NEB ligase buffer (10x)
 * 2 μL oligo (oFABs 1362, 1263, 1361, and 980)
 * 15 μL DH2O

In PCR machine
Removed the completed reaction tubes at the end of the day, and put them in my box in the -20°C freezer.
 * 37°C for 60 mins
 * 95°C for 3 mins
 * 4°C indefinitely


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