Team:UCLA/Notebook/Spider Silk Genetics/7 July 2015

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7/7/2015

PCR for ICA on M-270 Beads

• Using solution saved over from yesterday, performed PCR to assess ICA.
• Used two different concentrations of template due to low nanodrop reading.
  • Nanodrop indicated 4.12 ng/uL.
• Set up 2x 25 uL PCR reactions, used post-elution primers to amplify.

	1 uL of ICA Eluate	0.5 ng of ICA DNA (based on nanodrop)
5x Q5 buffer	5 uL	5 uL
10 mM dNTPs	0.5 uL	0.5 uL
10 uM For	1.25 uL	1.25 uL
10 uM Rev	1.25 uL	1.25 uL
5x GC Enhancer	5 uL	5 uL
Template	1 uL	1.2 uL (of a 1:10 dilution)
ddH20	10.75 uL	10.55 uL
Q5 Polymerase	0.25 uL	0.25 uL
Total	25 uL	25 uL

98 C	30 sec
98 C	10 sec
66 C	15 sec
72 C	15 sec
repeat from step 2	25 x
72 C	2 min
12 C	hold

Results

• Visualize results on 1% e-gel, using 0.5 uL 50 bp ladder.

Fig. 1 E-gel of post-elution primer amplification of ICA on M-270 beads. The expected size of the desired product is ~406 bp. In addition to the expected bands, there are bands present at ~100 bp and at ~750 bp.

• This indicates that we are able to get ICA working using M-270 beads. It seems that the problem we had previously was due to using streptavidin magnabind beads, which may be incompatible, or degraded.
• The bands at 100 bp are most likely due to the amplification of the IT construct, may be reduced in the future by using less initiator, compared to the 1 uL of 5 uM initiator used for this experiment.
• The bands above 400 bp may be due to over-amplification, and can be eliminated through optimizing the PCR.

PCA of MaSp2 Sequencing Core

• We are adding the extension primers to the MaSp2 Sequencing Core.
• The core is at a concentration of 13.04 ng/uL
  • We are using a 1:1000 dilution of the sequencing core as template: 13 pg/uL
• We only need to make MaSp2 Seq AB.
• NEB Tm Calculator gives 62 C as annealing temperature based on the overlap region. We decide to test 61 C and 64 C.

	Volume (uL)
5x Q5 buffer	5 uL
10 nM dNTPs	0.5 uL
10 uM For (2 A-F)	1.25 uL
10 uM Rev (2 B-R)	1.25 uL
MaSp2 Seq. Core (10 pg)	0.8 uL
5x GC enhancer	5 uL
ddH20	10.95 uL
Q5 Polymerase	.0.25 uL
Total	25 uL

98 C	30 sec
98 C	10 sec
61, 64 C	15 sec
72 C	10 sec
repeat from step 2	25 x
72 C	2 min
12 C	hold

• Saved PCR in -20 C.