Team:UCLA/Notebook/Spider Silk Genetics/3 August 2015

iGEM UCLA




8/3/2015

Gel Purification for M1-AB, BC, CA

  • Used zymo kit.
  • All around 25 ng/uL concentration, eluted in 15 uL.

PCA for M1-Seq1, M1-Seq2 Cores

  • M1-Seq1
    • Calculated 67 C annealing for overlap region
    • Test 65 C, 68 C.
  • M1-Seq2
    • Calculated 63 C annealing for overlap region
    • Test 62 C, 64 C.
  • Set up 2x 25 uL reactions for each M1-Seq core.
Volume (uL)
5x Q5 Buffer 5
d10 mM dNTPs 0.5
10 uM For 1.25
10 uM Rev 1.25
ddH20 16.75
Q5 Polymerase 0.25
Total 25
98 C 30 sec
98 C 10 sec
Anneal 15 sec
72 C 10 sec
repeat from step 2 25x
72 C 2 min
12 C hold

Results

Fig. 1 PCA of the M1-Seq Cores. The expected product size is 80 bp. The product for M1-Seq1 exhibits accessory bands. The product for M1-Seq2 appears mostly clean.
  • The proper bands were excised, then gel purified.

Digestion for M1-9, M2-15(1C3)

  • Digestion was in 50 uL reactions, with 1 uL of the appropriate enzymes.
Digestion Amount Enzymes Post-Digestion Purification
M2-15(1C3) 3 ug XbaI, PstI Gel purify
M1-9 all (~250 ng) EcoRI, PstI PCR purify
  • Digest at 37 C for 1.5 hrs, heat kill at 65 C for 20 min.
  • Run results on 1% TAE gel, used 2 uL of NEB 1kb ladder.
Fig. 2 Digestion of M2-15(1C3) with XbaI, PstI for subcloning into BBa_K525998. The expected product size is 1.6 kb. This band was excised for gel extraction.

ICA for M1-12

  • Incubated initiator for 1 hr.
  • Used 100 uL washes.
  • 3 minute ligation times.
  • Elute in 15 uL of 0.01% Tween-20.

Ligation

  • Ligated M2-15(X,P) into BBa_K525998(S,P)
  • Ligated M1-9(E,P) into pSB1C3(E,P).
  • 20 uL reactions.
  • Ligate 25 C for 1 hr, heat kill at 65 C for 20 min.

Transformation

  • Transform M2-15(T7) into BL21(DE3) chemically competent cells.
  • Transform M1-9(1C3) into DH5(alpha) electro-competent cells.