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Team:Harvard/allergy/notebook
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''amiRNA'' | ''amiRNA'' | ||
* amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21) | * amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21) | ||
| - | + | **ligations of Bet/LTP into V0120 | |
''ihpRNA'' | ''ihpRNA'' | ||
| - | + | * no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer) | |
| - | + | * redoing ligations of ihpRNA inserts in V9/V10 | |
**Redigested and gel purified some backbone | **Redigested and gel purified some backbone | ||
Revision as of 02:41, 24 October 2010
notebook calendar
06-14-2010 [ top ]
06-15-2010 [ top ]
06-16-2010 [ top ]
06-17-2010 [ top ]
06-18-2010 [ top ]
06-21-2010 [ top ]
- PCR amplification of gDNA from Arabadopsis thaliana for sense and antisense parts of LTP, Bet v 1, and Ger3.
- Diagnostic Digest of PCR products
Results
Lane 2 is LTP sense, lanes 11 and 12 are Ger 3 sense and antisense.
06-22-2010 [ top ]
- Gel electrophoresis of PCR products
- Gel digest and purification of LTP sense, Ger sense and Ger antisense
- Digest with Xba and Pst
- Purification of parts
Results
Gel purification yielded 2.6, 7.8, and 7.9 ng/μL of LTP sense, Ger sense, and Ger antisense parts. We were able to obtain 1.5, 4.3 and 0.5 ng/μL of digested LTP sense, Ger 3 sense, and Ger 3 antisense, respectively, from the digest purification.
06-23-2010 [ top ]
- Digest and purification of vector V0120
- Ligation of LTP sense, Ger sense, and Ger antisense parts into V0120
Results
Ligations were plated.
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
08-06-2010 [ top ]
amiRNA
- amiRNA-transformed turbo cells didn't grow in YEB medium. There should be more LB+Amp available by the end of the day. In the meantime, we're digesting some more backbone (8x20ul X+P+B21)
- ligations of Bet/LTP into V0120
ihpRNA
- no colonies on plates from transformations done yesterday after 16 hours (we let these plates grow for longer)
- redoing ligations of ihpRNA inserts in V9/V10
- Redigested and gel purified some backbone
- Concentrations: V9 (5 ng/uL); V10 (6 ng/uL)
- 12 ligations each for V9 and V10
