"
Team:Harvard/allergy/notebook
From 2010.igem.org
(Difference between revisions)
(→08-05-2010 [ top ]) |
(→08-05-2010 [ top ]) |
||
| Line 169: | Line 169: | ||
[[Image:V9v10.jpg|240px]] | [[Image:V9v10.jpg|240px]] | ||
| - | Lanes: Ladder, V9, V10 | + | Lanes: Ladder, V9, Ladder, V10 |
''Concentrations'': V9 (9.4 ng/uL; V10 (16.4 ng/uL) | ''Concentrations'': V9 (9.4 ng/uL; V10 (16.4 ng/uL) | ||
| Line 177: | Line 177: | ||
[[Image:AmiRNA-insert-diagnostic.jpg|240px]] | [[Image:AmiRNA-insert-diagnostic.jpg|240px]] | ||
| - | Lanes: | + | Lanes: 1-7: Bet, corresponding to 65.55 degrees C for stitching Tm (even spacing) |
| - | Lanes: 8- | + | Lanes: 8-10: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing |
* Digested | * Digested | ||
Revision as of 20:42, 22 October 2010
notebook calendar
06-14-2010 [ top ]
- Task #1
- Task #2
- Task #3
Image of gel, photo of your work, diagram, etc.
Results/ Conclusions
- We successfully cloned x, y, z
- Next step is to ligate 1, 2, 3
06-15-2010 [ top ]
06-16-2010 [ top ]
06-17-2010 [ top ]
06-18-2010 [ top ]
06-21-2010 [ top ]
06-22-2010 [ top ]
06-23-2010 [ top ]
06-24-2010 [ top ]
06-25-2010 [ top ]
06-28-2010 [ top ]
06-29-2010 [ top ]
06-30-2010 [ top ]
07-01-2010 [ top ]
07-02-2010 [ top ]
07-05-2010 [ top ]
07-06-2010 [ top ]
07-07-2010 [ top ]
07-08-2010 [ top ]
07-09-2010 [ top ]
07-12-2010 [ top ]
07-13-2010 [ top ]
07-14-2010 [ top ]
07-15-2010 [ top ]
07-16-2010 [ top ]
07-19-2010 [ top ]
07-20-2010 [ top ]
07-21-2010 [ top ]
07-22-2010 [ top ]
07-23-2010 [ top ]
07-26-2010 [ top ]
07-27-2010 [ top ]
07-28-2010 [ top ]
07-29-2010 [ top ]
07-30-2010 [ top ]
08-02-2010 [ top ]
08-03-2010 [ top ]
- Grew up cultures of completed ihpRNA constructs (Bet, LTP, Ger) in pORE expression vector
- amiRNA PCR
- Will look at results of PCR tommorrow
08-04-2010 [ top ]
Tasks
- amiRNA PCR appears to have worked at every Tm we tried:
- Digested V9/V10 to insert our constructs into
- Realized that we hadn't gel purified our ihpRNA inserts
- Gel purification of inserts (entire ihpRNA parts)
Ladder, 9, 11c1, 11c2, ladder, 25c1, 28c1, 28c2, 36c1, 36c2, ladder
Results
- Successfully gel purified our inserts and digested backbones that we will ligate into
08-05-2010 [ top ]
- Gel extracted V9/V10 backbone
Lanes: Ladder, V9, Ladder, V10
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:
Lanes: 1-7: Bet, corresponding to 65.55 degrees C for stitching Tm (even spacing)
Lanes: 8-10: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing
- Digested
- Bet,LTP inserts with X+P; B21 with X+P+phosphatase
- Ligated, Transformed
