Team:UCLA/Notebook/Spider Silk Genetics/1 July 2015

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7/1/2015

Initiator Binding Test

• Test initiator binding only.
  • Bind, then elute, then analyze on e-gel.
• Test four conditions:

1. Init only
2. Init + beads, then elute
3. Init + A + B
4. Init + term.

• 1 uL of 5 uM initiator used, 1 uL of 5 uM terminator.
• 50 ng of each other fragments.
• The same protocol as 6/19/2015 was used, with the following modifications.
  • initial binding time was increased to 45 minutes.
  • Washes were performed using 100 uL of solution.

Results

• After elution, we visualized the constructs on 1% e-gel.
• Nothing showed up on the gel, although we expect the initiator in condition 1 to appear.
  • Possibly due to mistakes made during sample preparation.

BsaI Digestion

• Set up 2x 50 uL reactions for each MaSp monomer.
• Digested ~5 ug of each MaSp plasmid using 4 uL of BsaI.
• Incubated 2 hours at 50 C, then heat for 20 minutes at 65 C.
• Visualized results on 1.5% gel, using 2 uL of 50 bp ladder.

Fig. 1 BsaI digestion of MaSp plasmids. The desired product is 102 bp in size.

• We excised the bands for purification tomorrow.

MaSp 2 6-mer ligation

• Tested 6-mer creation without use of beads.
• Experimentally create: IAB--CA--BCT, then pool all three sub-constructs together.
• Each sub-construct was ligated for 10 minutes at 25 C, then all three were pooled together, then incubated for another 10 minutes at 25 C.

	IAB	CA	BCT
DNA 1	1 uL	2.11 uL	1.74 uL
DNA 2	1.67 uL	1.67 uL	2.11 uL
DNA 3	1.74 uL	n/a	1 uL
2x T7 Ligase Buffer	5 uL	5 uL	5 uL
T7 Ligase	0.5 uL	0.5 uL	0.5 uL
ddH2O	0.09 uL	0.72 uL	-0.35 uL*
Total	10 uL	10 uL	10.35 uL

• *The reaction volume for BCT slightly exceeded 10 uL.

Results

• Ran the ligation product on 1% TAE gel, using 2 uL 100 bp ladder.

Fig. 2 Ligation of MaSp2 6-mer. The expected product for a 6-mer ligation is ~712 bp. There are other bands present that may be due to other possible ligation events.

• We successfully created a 6-mer ligation product without beads, although the results is far from efficient due to the presence of a number of bands that correspond to other ligation events.
• We excised the 712 bp band for gel extraction tomorrow.
• We plan to PCR amplify the 6-mer and ligate it into pSB1C3.
• This result indicates that successive ligation can only be perfomed twice, as ligation efficiency decreases dramatically. This means that for creating larger MaSp constructs such as 12- or 15-mers, we would need successive ligation and purification steps to achieve efficient construction.