Team:UCLA/Notebook/Spider Silk Genetics/7 August 2015
Contents
8/7/2015
Redid PCR for M1-12mer
| 5X Q5 buffer | 10 uL |
| 10mM dNTPs | 1 uL |
| F-03 | 2.5 uL |
| G-03 | 2.5 uL |
| Template | 1 uL |
| ddH2O | 32.5 uL |
| Q5 Polymerase | 0.5 uL |
| 98 | 30 sec |
| 98 | 10 sec |
| 66 | 20 sec |
| 72 | 35 sec |
| x23 | |
| 72 | 2 min |
| 12 | Hold |
BsaI Digestion M1 constructs
| M1 AB (554.88 ng/uL) | M1 BC (1477 ng/uL) | M1 CA (648 ng/uL) | |
| Cutsmart | 5 uL | 5 uL | 5 uL |
| Plasmid (5 ug) | 9 uL | 3.4 uL | 7.7 uL |
| ddH2O | 32 uL | 37.6 uL | 33.3 uL |
| BsaI | 4 uL | 4 uL |
| 50C | 2hr |
| 65C | 20 min |
EcoRI + PstI Digestion of M1SeqAB 1 and 2
| M1SeqAB1 (128 ng/uL) | M1SeqAB2 (222ng/uL) | |
| EcoRI | 1 uL | 1 uL |
| PstI | 1 uL | 1 uL |
| Buffer 2.1 | 5 uL | 5 uL |
| DNA | 7.8 uL | 4.5 uL |
| ddH2O | 35.2 uL |
| 37C | 1.5 hr |
| 65C | 20 min |
XbaI + Pst1 Digestion of M1-9 (seq verified plasmid 1)
| M1-9mer plasmid (454 ng/uL) | |
|---|---|
| XbaI | 1 uL |
| PstI | 1 uL |
| 10X Buffer 2.1 | 5 uL |
| DNA (3ug) | 6.6 uL |
| ddH2O | 36.4 uL |
| 37C | 1.5 hr |
| 65C | 20 min |