Minnesota/24 July 2008

From 2008.igem.org

(Difference between revisions)
Line 46: Line 46:
| BV || 1.0uL || 5.1uL
| BV || 1.0uL || 5.1uL
|}
|}
 +
 +
|-
 +
|'''5. Gel electrophoresis:''' After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.
 +
|-
 +
|'''6. Gel Purified:''' Cut out bands from gel and purified those bands (which contain the DNA wanted).
 +
|-
 +
|'''7. Spetrophotometry:''' Spec the purified gel DNA to measure concentration of DNA in eight parts.

Revision as of 21:41, 24 July 2008

Back to Notebook Home
Go to Previous Day (July 23)Go to Next Day (July 25)
1. Base Vector Plasmid Maxi Prep Base Vector: Grew another culture of different base vector colonies, so now need to plasmid prep the base vector so the DNA/plasmid will be extracted from the rest of the cell.
2. Spectrophotometry: Excellent results from spec'ing new base vector plasmid. Very high concentration of DNA, which was 430 ng/uL.
3. Base Vector & Promoter Double Digest: 50uL Reaction Mixture. Digest incubation for 2 hours @ 37C. Heat inactivate enzyme for 15 minutes @ 65C in a water bath. Follow the table below for pre-incubation steps:


Parts 10x Buffer BSA H20 DNA from parts RE 1 RE 2 [DNA] ng/uL
BV 5.0uL 0.5uL 30.8uL 11.7uL EcoRI, 1.0uL Pst1, 1.0uL 100.62 ng/uL
TetR Pro. 1 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL
Tet R Pro. 2 5.0uL 0.5uL 35.5uL 7.0uL EcoRI, 1.0uL Pst1, 1.0uL 23.8 ng/uL
LacI Pro. 1 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL
LacI Pro. 2 5.0uL 0.5uL 41.5uL 1.0uL EcoRI, 1.0uL Pst1, 1.0uL 168.0 ng/uL
Lac/Lam 1 5.0uL 0.5uL 28.5uL 14.0uL EcoRI, 1.0uL Pst1, 1.0uL 9.8 ng/uL
Tet/p22 1 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL
Tet/p22 2 5.0uL 0.5uL 34.0uL 8.5uL EcoRI, 1.0uL Pst1, 1.0uL 20.4 ng/uL
4. Vector Dephosphorylation: After the digested products' enzyme has been heat inactivated, we dephosphorylated the base vector so the cut ends wouldn't religate. Follow the table below:


Parts Antarctic Phosphatase Antarctic Phosphatase Buffer
BV 1.0uL 5.1uL
5. Gel electrophoresis: After heat inactivating the enzyme @ 65C for 15 minutes, we ran a gel with the BV and double digest proucts.
6. Gel Purified: Cut out bands from gel and purified those bands (which contain the DNA wanted).
7. Spetrophotometry: Spec the purified gel DNA to measure concentration of DNA in eight parts.
Retrieved from "http://2008.igem.org/Minnesota/24_July_2008"