Team:UCLA/Notebook/Spider Silk Genetics/20 July 2015

iGEM UCLA




7/20/2015

BsaI Digestion of MaSp2 AB, BC, CA

Sub-cloning M2-9(1C3) into BBa_K525998: Preparation

  • Digest 10 uL of plasmid (~2.8 ug) using 1 uL XbaI and 1 uL of PstI in a 50 uL reaction.
    • Used NEBuffer 2.1
  • Digest 37 C for 1.5 hrs, heat kill 65 C for 20 min.
  • Gel-purify resulting product.

Cloning M2-12 into pSB1C3: Preparation

  • Digest 15 uL PCR product using 1 uL each of EcoRI and PstI in a 50 uL reaction
    • Used NEBuffer 2.1
  • Digest 37 C for 1.5 hrs, heat kill 65 C for 20 min.
  • Gel-purify resulting product.

Gel Purification Results

  • Cast 1.5% TAE gel. Used 2 uL each of 100 bp ladder and 1 kb ladder from NEB.
Fig. 1 Gel image of results of BsaI Digestion, M2-9(1C3) digestion, and M2-12 digestion. The expected size for BsaI digestion is 102 bp. The expected size for M2-9 is 1018 bp. The expected size for M2-12 is 1324 bp.
  • Excised all the above indicated bands for gel purification.

Ligation

  • M2-9(1C3) into BBa_K525998, and M2-12 into pSB1C3.
  • Used [insilico.uni-duesseldorf.de ligation calculator] to determine amounts of DNA to add.
    • vector size: 2200 bp.
    • vector amount: 50 ng.
    • insert size:
      • M2-9: 1018 bp
      • M2-12: 11224 bp
    • vector to insert ration: 1 to 3
  • Calculated for 50 ng of vector, add:
    • 69.41 ng for M2-9
    • 83.45 ng for M2-12.
  • Ligation in 20 uL volumes.
    • Ligate 25 C for 25 min, heat kill 65 C for 10 min.

Transformation

  • Transformed M2-9(T7) into chemically competent BL21(DE3) cells.
  • Transformed M2-12(1C3) into chemically competent BL21(DE3) cells.
    • Previous tranformations of M2-mers(1C3) were performed using DH5(alpha) cells.
  • Rescues were at 37 C for 30 min.

Glycerol Stock Reconstitution

  • Plated M2-AB, M2-BC, M2-CA, and M2-SeqAB from glycerol stocks.
  • Aim to grow culture for DNA prep.

PCA of MaSp1 Temperature Testing

  • Testing annealing temperature for end-extension of the MaSp1 sequence.
  • NEB Tm calculator gives 64 C as ideal annealing temperature.
  • Used the following protocol.
Volume (uL)
5x Q5 Buffer 5
M1-AF 1.25
M1-BR 1.25
dNTPs 0.5
Template (10 pg) 0.6
ddH20 16.15
Q5 Polymerase 0.25
Total 25
98 C 30 sec
98 C 10 sec
66/64 C 15 sec
72 C 15 sec
repeat from step 2 25x
72 C 2 min
12 C hold

Results

  • Cast 1.5% TAE gel to visualize. Used 3 uL of NEB 50 bp ladder.
Fig. 2 Temperature testing for PCR end-extension of MaSp1 core. The expected product is 170 bp.
  • Both temperatures work well, however, 64 C has less non-specific amplification. We will use 64 C to anneal tomorrow.