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Team:Harvard/allergy/notebook
From 2010.igem.org
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, and Ger.
- 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 ]
- Digest and gel electrophoresis of PCR products
- Gel digest and purification of LTP sense, Ger sense and Ger antisense
Results Obtained 2.6, 7.8, and 7.9 nanograms per microliter of LTP sense, Ger sense, and Ger antisense parts.
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
