Team:UCLA/Notebook/Spider Silk Genetics/24 July 2015

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7/24/2015

Sequencing Results

• M1-AB
  • All three good.
• M1-BC
  • All three good.
• M1-CA
  • All three good.
• M2-12(1C3)
  • 1: No insert
  • 2: No insert
  • 3: Gibberish (does not align to anything)
• Unfortunately, no correct sequences for M2-12(1C3)
  • Maybe re-transform using first ligation product.

PCR Amplification for M-12

• Amplify using post-elution primers in 1x 50 uL reaction.

	Volume (uL)
5x Q5 Buffer	10
10 mM dNTPs	1
10 uM For (F-03)	2.5
10 uM Rev (G-03)	2.5
Template	1
Q5 Polymerase	0.5
ddH2O	32.5
Total	50 uL

98 C	30 sec
98 C	10 sec
66 C	20 sec
72 C	40 sec
repeat from step 2	20x
72 C	2 min
12 C	hold

• After PCR, sample was split into two equal volumes for gel purification.
• Verified on 1% TAE gel, with 2 uL of NEB 1kb ladder. (Fasih loaded this gel, and was somewhat rough with it.).

Fig. 1 PCR amplification of ICA M2-15mer. The expected size for M2-15 is 1630 bp. The two lanes marked M2-15 are identical samples. The two unmarked lanes have no sample in them.

• Not a lot of yield this time. May need to PCR more in the future.
• The 1630 bp band was excised, and gel purified using Qiagen kit.

Colony PCR for M2-12(1C3)

• Picked eight colonies from the plate of M2-12(1C3) retrieved 7/22/2015.
• Suspended colony in 15 uL LB.
• Streaked pipet tip after resuspension on an LB-Chloramphenicol index plate.

	Volume (uL)
2x Taq Red	12.5
10 uM For (VF)	1.25
10 uM Rev (VR)	1.25
Template (1:50)	1 uL
ddH2O	9 uL
Total	25 uL

95 C	3 min
95 C	25 sec
56 C	30 sec
72 C	45 sec
repeat from step 2	30x
72 C	5 min
12 C	hold

• Negative control colony PCR: instead of colony, used 1 uL of plain LB.

Results

• Visualized colony PCR on 1% TAE gel, using 3 uL of NEB 1kb ladder.

Fig. 2 Colony PCR of M2-12(1C3) The expected size is approx 1.6 kb, since this was amplified using VF/R. None of the bands are close to 1.6 kb.

• Colony PCR indicates that all of the colonies we tested do not have the desired insert. This is likely symptomatic of the entire plate.
• We will abandon this plate of bacteria, since it most likely does not contain any usable colonies.