Team:Harvard/allergy/notebook

(Difference between revisions)

notebook calendar

Week 1	06-14-2010	06-15-2010	06-16-2010	06-17-2010	06-18-2010
Week 2	06-21-2010	06-22-2010	06-23-2010	06-24-2010	06-25-2010
Week 3	06-28-2010	06-29-2010	06-30-2010	07-01-2010	07-02-2010
Week 4	07-05-2010	07-06-2010	07-07-2010	07-08-2010	07-09-2010
Week 5	07-12-2010	07-13-2010	07-14-2010	07-15-2010	07-16-2010
Week 6	07-19-2010	07-20-2010	07-21-2010	07-22-2010	07-23-2010
Week 7	07-26-2010	07-27-2010	07-28-2010	07-29-2010	07-30-2010
Week 8	08-02-2010	08-03-2010	08-04-2010	08-05-2010	08-06-2010
Week 9	08-09-2010	08-10-2010	08-11-2010	08-12-2010	08-13-2010

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

08-09-2010 [ top ]

08-10-2010 [ top ]

08-11-2010 [ top ]

08-12-2010 [ top ]

08-13-2010 [ top ]