Team:UCLA/Notebook/Spider Silk Genetics/13 July 2015

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7/13/2015

BsaI Digestion of MaSp2 Plasmids and Gel Purification

• digested 5 ug of MaSp2 AB, BC, CA each using 4 uL of BsaI in 50 uL reactions.
  • 2x 50 uL reactions for each

	AB (112 ng/uL)	BC (205 ng/uL)	CA (219 ng/uL)
10x Cutsmart	5 uL	5 uL	5 uL
DNA	44 uL	25 uL	23 uL
ddH2O	-3 uL	16 uL	18 uL
BsaI	4 uL	4 uL	4 uL
Total	50 uL	50 uL	50 uL

• Incubate 50 C for 2 hrs, then 65 C for 20 min.
• Ran samples on 1.5% TAE gel, with 2 uL of 100 bp ladder to visualize.

Fig. 1BsaI Digestion of MaSp2 plasmids. The expected product is at 102 bp. These 102 bp bands were excised for gel extraction.

• Gel extracted the indicated bands using Qiagen kit. Yields were approximately 20 ng/uL in 11 uL of ddH2O.

PCR Amplification of ICA 6- and 9-mer

• Using the ICA eluate from 7/10/2015 for the template.
• Conducted a temperature test to determine proper annealing for best amplification.
• We also decided not to use GC enhancer in this protocol to see how it turns out.

	Volume (uL)
Q5 Rxn buffer	5 uL
F-03 primer	1.25 uL
G-03 primer	1.25 uL
dNTPs	0.5 uL
Template (6- or 9-mer)	0.5 uL
ddH2O	16.25 uL
Polymerase	0.25 uL

98C	5 uL
98C	1.25 uL
66/ 68.5C	1.25 uL
72C	0.5 uL
Repeat	x25
72	2 min
12	Hold

• Cast 1.5% TAE gel to visualize, used 2 uL of 100 bp ladder.

Fig. 2 Temperature test for amplifying 6-mer and 9-mer ICA. The expected product for 6-mer is 712 bp. The expected product from 9-mer is 1018 bp. The samples indicated as "Old" were amplified at 66 C annealing, with GC enhancer used.

• It seems that 6-mer amplification is somewhat successful. There is the desired band present, although there are also non-specific bands present.
• 9-mer amplification seems to be inefficient. The desired band is present, but is very weak intensity compared to 6-mer. In addition, non-specific bands are present.
• We don't know is the accessory bands are an artifact from ICA, or an artifact from PCR. One way to verify would be to excise the band corresponding to the desired product, gel-extract, then use as template for a subsequent PCR. If the accessory bands reappear, then the problem lies with non-specific primers. If the bands are no longer present, then the artifact bands arise from a problem with ICA.