IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/22
|iGEM iGarden||Main project page|
Previous entry Next entry
Today's objective is to take our PCR products from yesterday (LTP sense, Ger 3 sense, and Ger 3 antisense)and insert them into vector V0120 for E. coli. Our procedures were:
1. Gel Extraction of LTP sense, Ger 3 sense, and Ger 3 antisense 2. Purification of LTP sense, Ger 3 sense, and Ger 3 antisense
Obtained 2.6, 7.8 and 7.9 ng/μL of LTP sense, Ger 3 sense, and Ger 3 antisense, respectively, after ourgGel extraction and purification. These are relatively small concentrations. These tubes of DNA will be digested and then purified again.
3. Digest of LTP sense, Ger 3 sense, and Ger 3 antisense with Xba and Pst restriction enzyme
4. Purification of digested allergens LTP sense, Ger 3 sense, and Ger 3 antisense
Obtained 1.5, 4.3 and 0.5 ng/μL of digested LTP sense, Ger 3 sense, and Ger 3 antisense, respectively. The drop in concentration is attributed to loss of DNA through the purification process.
5. Digest of V0120 with Xba and Pst restriction enzymes 6. Gel Electrophoresis of the digested vector
The properly digested vector is 3000bps long, and rests between two bands on the gel -- the uncut or one cut vector and the removed inserts.
7. Gel Extraction and Purification of Vector Nanodrop of digested vector yielded 25.2 ng/μL.
8. Ligation of V0120 and inserts for each LTP sense, Ger 3 sense, and Ger 3 antisense
Ligated vectors were put on ice and set aside for tomorrow, where they will be transformed into competent turbo E. coli, which will be grown in plates of agar. Colonies that grow should contain closed vector plasmid, which contains ampicillin resistance and inserts.
PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)
1=95°C for 10:00
2=95°C for 00:15
3=50°C for 00:30
4=72°C for 01:30
5=steps 2-4 X 29
6=72°C for 10:00
7=4°C for ∞
PCR for E10 LacI
File:Cutout622.jpg To Make Agarose Gel
Colonies from the following cultures were innoculated and set to shake at 37°C overnight: