Team:UCLA/Notebook/Spider Silk Genetics/7 May 2015
Performed a Ligation/Digestion test based on golden gate cloning, reactions as follows:
Tube 1 Tube 2 (control)
2AB (380 ng/uL) 2.63 uL 0 uL
2BC (460 ng/uL) 2.17 uL 4.34 uL
10x T4 Ligase buffer 2 uL 2 uL
BSA (1mg/mL) 2 uL 2 uL
BsaI (15 U) 1.5 uL 1.5 uL
T4 Ligase (5 U)* 1.25 uL 1.25 uL
ddH20 8.45 uL 8.91 uL
Total 20 uL 20 uL
37 ℃ 20 s
37 ℃ 2 min
*
16 ℃ 3 min
50 ℃ 5 min
*Repeat from * 30 x
80 ℃ 5 min
12 ℃ hold
*T4 Ligase was diluted 1:100 in 1x T4 Ligase buffer prior to adding 1.25 uL
1 ug of each plasmid was used.
Visualized Ligation/Digestion Test on 2% TAE gel. Results indicate that digestion occurred ~50% efficiency. The undigested and digested backbones appeared at approx. equal intensity. No bands smaller than 1000 bp appeared, cannot say for sure whether ligation took place.
Gel purified BsaI digested plasmids from yesterday. Yields: ~7 ng/uL for AB, BC, CA.