Team:UCLA/Notebook/Spider Silk Genetics/16 July 2015

iGEM UCLA




7/16/2015

Transformation Results

  • MaSp2 3-, 6-mer in BBa_k525998 in BL21 cells have many colonies on the plate.
    • May have plated too much. Will reduce plating density in the future when working with the BL21(DE3) chemical competent cells.
  • 9-mer in pSB1C3 has very few small colonies.

MaSp2 ICA 12-mer

  • Plan to do 12 mer today: 100 uL wases, 5 min ligation time.
  • 1 ul of 50 nM initiator used. Initial binding in 45 minutes.
  • 5 minute ligation times for each step.
  • 50 ng of each DNA species
  • 1 uL of 5 uM solution of each relevant cap.
  • eluted in 15 uL of 0.01% Tween-20 solution

PCR amplification for MASp2 ICA 12-mer

  • Used the following protocol to set up 2x 25 uL reactions.
  • Used crude eluate from ICA as template.
  • Amplified with post-elution primers
Volume (uL)
5x Q5 Buffer 5
F-03 1.25
G-03 1.25
dNTPs 0.5
Template 0.5
ddH2O 16.25
Q5 Polymerase 0.25
Total 25
98 C 30 sec
98 C 10 sec
66 C 20 sec
72 C 35 sec
repeat from step 2 20 x
72 C 2 min
12 C hold

Results

  • Cast 1.5% TAE gel to visualize results; used 2 uL of NEB 100 bp ladder.
Fig. 1 PCR Amplification of MaSp2 ICA 12-mer using post-elution primers. Both lanes are identical conditions. The expected product is 1324 bp. This is the major band present. Slight accessory bands are also present, but are a minor product.
  • We were able to successfully perform ICA to obtain a 12-mer. The accessory bands present may be due to PCR non-specific amplification, or to inaccuracies in the ICA process.
  • We excised the 1324 bp band for gel-extraction in preparation for cloning.

Liquid Culture for MaSp2 3-mer, 6-mer, 9-mer

  • Grew 3x 5 mL culture for MaSp2 3-mer(T7), MaSp2 6-mer(T7), and 9-mer(pSB1C3).
  • Plan to miniprep tomorrow then sequence over the weekend.
  • Will have bacterial cultures ready on Monday for the protein expression team to use.