Team:Harvard/allergy/notebook

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* Digest and gel electrophoresis of PCR products
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* Gel electrophoresis of PCR products
* Gel digest and purification of LTP sense, Ger sense and Ger antisense
* Gel digest and purification of LTP sense, Ger sense and Ger antisense

Revision as of 22:15, 22 October 2010



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, and Ger.
  • Diagnostic Digest of PCR products

Results

6.17.jpg

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

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:

AmiRNA-try-3.jpgAmiRNA pcr 8.3.jpg


  • 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)

Gel 1

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

V9v10.jpg

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:

AmiRNA-insert-diagnostic.jpg

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 ]

08-09-2010 [ top ]

08-10-2010 [ top ]

08-11-2010 [ top ]

08-12-2010 [ top ]

08-13-2010 [ top ]