Team:UCLA/Notebook/Spider Silk Genetics/21 April 2015

iGEM UCLA




4/21/2015

4/21/2015


1 Ligation of MaSp 2AB, 2BC, 2CA in to pSB1C3

∙ Used ligation calculator to determine amounts of each insert to use to ligate into 50 ng of vector for a 3:1 ratio of insert to vector.

∙ 2000 bp vector, 170 bp insert.

∙ Determined 12.75 ng of insert to 50 ng vector. Proceeded with ligation using protocol below:

2AB (43.28 ng/uL)2BC (34.56 ng/uL)2CA (34.9 ng/uL)
T4 Ligase 1 uL 1 uL 1 uL
Insert (12.75 ng) 0.30 uL 0.370 uL 0.365 uL
Vector (50 ng) 1.484 uL 1.484 uL 1.484 uL
10x T4 Ligase Buffer2 uL 2 uL 2 uL
ddH2O 15.22 uL 15.15 uL 15.16 uL
Total 20 uL 20 uL 20 uL

25 C20 min
65 C 10 min

2 Bacterial Transformation

∙ Used 2 uL of each reaction to transform 50 uL competent cells.

∙ Incubate on ice 5 min.

∙ Add 200 uL of pre-warmed SOC to each tube.

∙ Rescue for 10 min.

∙ Plate 50 uL onto chloramphenicol plates. Incubate at 37 C overnight.

3 MaSp2 PCA

∙ Set up 2x 50 uL reactions for AB, BC, CA each using protocol from 4/13/2015 scaled to 50 uL with the following modifications:

∙ Annealing temp: 64 C

∙ 25 cycles instead of 20.