Team:Harvard/allergy/notebook

From 2010.igem.org

(Difference between revisions)
(06-21-2010 [ top ])
(08-06-2010 [ top ])
Line 200: Line 200:
==08-06-2010 [ [[#top|top]] ]==
==08-06-2010 [ [[#top|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|top]] ]==
==08-09-2010 [ [[#top|top]] ]==

Revision as of 02:39, 24 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 v 1, and Ger3.
  • 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
  • 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:

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 ]

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 ]