Team:UCLA/Notebook/Spider Silk Genetics/12 August 2015

iGEM UCLA




08/12/2015

Colony PCR for M1 SwqAB 1 & 2 and M1-9(T7)

2X Taq MM 6.25 uL
VF Primer 0.625 uL
VR Primer 0.625 uL
Template 1 uL
ddH2O 4 uL
95C 3 min
95C 30 sec
72C 25 sec (M1SeqAB)/ 1min 40 sec (M1-9)
x30
72C 5 min
12C Hold
Lane 3-6: M1-9(T7) Colony 1-4. Lane 7-10: M1SeqAB1 Colony 1-4. Lane 11-14: M1SeqAB2 Colony 1-4

Grew liquid culture for

  • all 4 colonies of M1-9 (T7); they all showed band close to the expected size ~1200-1300bp but they are slightly different so decided to sequence all of them.
  • colonies 1-3 for M1SeqAB1
  • none for M1SeqAB2 since we probably do not need this seq construct

BsaI Digestion of M1AB, BC and CA

1AB (553.88 ng/uL) 1BC (1477.18 ng/uL) 1CA (648 ng/ uL)
Custmart 5 uL 5 uL 5 uL
DNA 7.2 uL 2.7 uL 6.2 uL
ddH2O 33.8 uL 38.3 uL 34.8 uL
BsaI 4 uL 4 uL 4 uL

50C for 2 hr 65C for 20 min

BsaIDig0812.jpg

Gel purified samples