Team:UCLA/Notebook/Spider Silk Genetics/17 August 2015

iGEM UCLA




8/17/2015

Sequencing Results

  • M1-9(T7)
    • 1: good
    • 2: good
    • 3: BAD
    • 4: good
  • M1-Seq1-AB:
    • 1, 2, 3 are all good, but M1-Seq1-AB identified here is actually M1-Seq2-AB.

Colony PCR for M1-12(1C3)

  • Picked 7 colonies, ran colony PCR using Apex 2x Taq Red in 12.5 uL reactions.
Volume (uL)
2x Taq Red 6.25
10 uM For (VF) 0.625
10 uM Rev (VR) 0.625
Colony 1
ddH2O 4
Total 12.5
95 C 3 min
95 C 25 sec
56 C 30 sec
72 C 1 min
repeat from step 2 30x
72 C 5 min
12 C hold
Fig. 1 Colony PCR of M1-12(1C3). The expected size for M1-12(1C3) is 1324 bp. The negative control exhibits a strong band. This may be due to contamination of any one of the stock ingredients.
  • ERRATA: The expected size for M1-12(1C3) when amplified using VF/R is 1.5 kb. All colonies present exhibit the band for M1-9(1C3) and M1-9(T7). This is most-likely due to cross contamination of supplies used during the transformation process.
  • ERRATA: The band present in the negative control is not due to the water used, or LB, as confirmed by Fasih, who performed colony PCR and negative control using LB. The prime candidates for this band are contamination in the primers or in the Taq Red, both of which are shared by all the members in iGEM.

BsaI Digestion for M1-AB, BC, CA and M2-AB, BC, CA

  • Digested 5 ug of each plasmid in 50 uL reactions with 4 uL of BsaI.
  • Digest for 2 hrs at 50 C, heat kill at 60 C for 20 min.
  • M1-AB, BC, CA were excised for subsequent purification.
  • M2-AB, BC, CA were NOT excised. The band pattern had a double band near the 100 bp mark, and the resulting digestion was unusable. The plasmid stocks used for digestion for M2-AB, BC, CA were dilute (~100 ng/uL), which may account for this result.
    • Plan to maxiprep more DNA from bacterial glycerol stocks.

M1-12(1C3) Liquid Culture

  • Grew 5 mL of colonies 1-4.
  • ERRATA: the colonies on the plate are contaminating M1-9(1C3) and M1-9(T7) colonies.