iGEM UCLA

8/30/2015

Gel Purification

Digested M1-12, M1/2[1:2]-12 each with E,P and X,P.
Digest 5 uL of the samples purified today in a 50 uL reaction with 1 uL each of the respective enzymes.
Digest 37 C for 1.5 hrs, heat at 65 C for 20 min.
Digests were purified using Zymo clean and concentrator.

For a 3:1 ligation to 50 ng of vector, 96 ng of 1324 bp insert is required
For a 5:1 ligation to 50 ng of vector, 160 ng of 1324 bp insert is required.
Ligated E,P digested products into pSB1C3.
Ligated X,P digested products into BBaK_525998.
For M1-12 ligations, used 10 uL of digested product to approximate a 3:1 insert:vector ratio due to insufficient amounts.
For M1/2[1:2]-12 ligations, used enough product to ligate at a 5:1 insert:vector ratio.
Ligate at 25 C for 1 hr, heat kill at 65 C for 20 min.

Transform M1-12(1C3) and M1/2[1:2]-12(1C3) into DH%(alpha) electrocompetent cells.
- Ligation products were dialyzed against ultra-pure ddH2O prior to transformation.
- Arc time of 5.6 ms for both.
Transform M1-12(T7) and M1/2[1:2]-12(T7) into chemically competent BL21(DE3) cells.

Set up 2x 11 mL starter culture of M1/2[1:1]-12(T7)and M1/2[2:1]-12(T7) each.
- One culture grown at 30 C, one grown at RT (~20 C)
Set up 11 uL of culture for M1-SeqAB2 (37 C).