User:Anthony Salvagno/Notebook/Research/2009/03/27/Plasmid Extraction and Digestion

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I'm in a quirky mood today and so... I want to mix it up if you would like.

Part 1

I followed a Qiagen miniprep protocol. It worked well. Last time I took cells from 6 of the 20 colonies and grew more cells. Then I followed this protocol to extract the plasmid DNA. Next I ran a nanodropper on each of the 6 different tubes (remember each tube should contain a different random fragment). My results:

Tube 1 - from culture 6 - 107.9 ng/ul Tube 2 - from culture 7 - 107.7 ng/ul Tube 3 - from culture 14 - 112.9 ng/ul Tube 4 - from culture 15 - 108.7 ng/ul Tube 5 - from culture 16 - 132.2 ng/ul Tube 6 - from culture 17 - 92.5 ng/ul

Good job me.

Part 2

Then I set up a digestion reaction. Here it is.

Reaction 1
Tube Number Contents Vol DNA Vol/# Buffer 10x Vol 10x BSA Vol XhoI Vol H2O Total Vol
1 pRS413 3.4uL #2, 1.5uL 1.5uL 1uL 7.6uL 15uL
2 Tube 1 4.6uL #2, 1.5uL 1.5uL 1uL 6.4uL 15uL
3 Tube 2 4.6uL #2, 1.5uL 1.5uL 1uL 6.4uL 15uL
4 Tube 3 4.4uL #2, 1.5uL 1.5uL 1uL 6.6uL 15uL
5 Tube 4 4.6uL #2, 1.5uL 1.5uL 1uL 6.4uL 15uL
6 Tube 5 3.8uL #2, 1.5uL 1.5uL 1uL 7.2uL 15uL
7 Tube 6 5.4 #2, 1.5uL 1.5uL 1uL 5.6uL 15uL
Reaction 2
Tube Number Contents Vol DNA Vol/# Buffer 10x Vol 10x BSA Vol BstxI Vol H2O Total Vol
1 pRS413 3.4uL #2, 1.5uL 1.5uL 1uL 7.6uL 15uL
2 Tube 1 4.6uL #2, 1.5uL 1.5uL 1uL 6.4uL 15uL
3 Tube 2 4.6uL #2, 1.5uL 1.5uL 1uL 6.4uL 15uL
4 Tube 3 4.4uL #2, 1.5uL 1.5uL 1uL 6.6uL 15uL
5 Tube 4 4.6uL #2, 1.5uL 1.5uL 1uL 6.4uL 15uL
6 Tube 5 3.8uL #2, 1.5uL 1.5uL 1uL 7.2uL 15uL
7 Tube 6 5.4 #2, 1.5uL 1.5uL 1uL 5.6uL 15uL

Part 3

Gel setup so we can see whether we got working ligations/insertions. Picture coming soon.

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