Team Fence
Colonies from yesterday's ligations
- applied 3x gel volume of QG buffer (in μL) to each respective tube
- placed in 42°C bath until the gel was melted
- added 1 gel volume of isopropanol to each, and vortexed
- applied contents of each tube to a QIAquick spin collumn
- spun for 1 min
- added 500μL QG buffer (to dissolve any remaining agarose)
- spun for 1 min
- added 750μL PE buffer
- spun for 1 min, discared flow-through
- spun for another minute
- placed QIAquick collumn in a fresh, labeled eppendorf tube, applied 50μL EB buffer to elute, and spun for 1 min
Nanodrop of Gal4 and EcR Gel Extractions
| Plasmid |
Quantity (ng/μL) |
260/280
|
| Gal-EcR (1)
|
3.1 |
2.45
|
| Gal-EcR (2)
|
4.5 |
1.72
|
| Gal-EcR (3)
|
3.0 |
11.56
|
| Gal-EcR (4)
|
3.6 |
2.21
|
Digestions
VP16
- 1μL xba
- 1μL Pst
- 5μL VP16(2) miniprep
- 1μL 10x FD green buffer
- 2μL DH2O
5xGalUAS
- 1μL xba
- 1μL Pst
- 2μL 5xGal4UAS (from Karmella)
- 1μL 10x FD green buffer
- 5μL DH2O
PhyB
- 1μL Bam
- 1μL Nco
- 16μL PhyB
- 2μL 10x FD green buffer
Team Flavor
Transformation of J45004
- 5μL of registry DNA was transformed with 15μL TURBO cells; mixture was plated on LB+AMP and left to grow O/N
Team Allergy
- Gel extracte V9/V10 backbone
Concentrations: V9 (9.4 ng/uL; V10 (16.4 ng/uL)
- Ligated ihpRNA inserts into V9/V10 and transformed
- For our ligations we only used ~ 2uL of backbone (around 18 and 32 ng of backbone)and used a 3x excess of insert
- Verified that amiRNA stitching of Bet, LTP yielded the proper insert with a low level of background through PCR:
- Digested Bet,LTP inserts with X+P, B21 with X+P+phosphatase
- Ligated, transformed
- 0-7: Bet, corresponding to 65..55 degrees C for stitching Tm (even spacing)
- 8-15: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing
|
iGEM iGarden
