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

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.