IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/05: Difference between revisions

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==Team Fence==
===Colonies from yesterday's ligations===
[[Image: 84 colonies 1.jpg]]
[[Image: 84 colonies 2.jpg]]
===Gel extraction of EcR-Gal Bam Nco===
*applied 3x gel volume of QG buffer (in μL) to each respective tube
*placed in 42°C bath until the gel was melted
*added 1 gel volume of isopropanol to each, and vortexed
*applied contents of each tube to a QIAquick spin collumn
*spun for 1 min
*added 500μL QG buffer (to dissolve any remaining agarose)
*spun for 1 min
*added 750μL PE buffer
*spun for 1 min, discared flow-through
*spun for another minute
*placed QIAquick collumn in a fresh, labeled eppendorf tube, applied 50μL EB buffer to elute, and spun for 1 min
===Nanodrop of Gal4 and EcR Gel Extractions===
{| border="3" style="margin-left: 3em;"
|-
! Plasmid !! Quantity (ng/μL) !! 260/280
|-
! Gal-EcR (1)
| 3.1 || 2.45
|-
! Gal-EcR (2)
| 4.5 || 1.72
|-
! Gal-EcR (3)
| 3.0 || 11.56
|-
! Gal-EcR (4)
| 3.6 || 2.21
|}
===Digestions===
VP16
*1μL xba
*1μL Pst
*5μL VP16(2) miniprep
*1μL 10x FD green buffer
*2μL DH<sub>2</sub>O
5xGalUAS
*1μL xba
*1μL Pst
*2μL 5xGal4UAS (from Karmella)
*1μL 10x FD green buffer
*5μL DH<sub>2</sub>O
PhyB
*1μL Bam
*1μL Nco
*16μL PhyB
*2μL 10x FD green buffer
===Sending EcR-Gal for sequencing===
*5μL of each primer, either fwd or rev, at 1/20 dilution
*10μL of sample and DH<sub>2</sub>O, such that sample is at 500ng total mass
Sealed tubes with parafilm.
Submited Gal-EcR 1, 7, 10, and 12 (the one's with inserts on the digestion gel from 8/4)
==Team Flavor==
==Team Flavor==
===Transformation of J45004===
===Transformation of J45004===
*5μL of registry DNA was transformed with 15μL TURBO cells; mixture was plated on LB+AMP and left to grow O/N
*5μL of registry DNA was transformed with 15μL TURBO cells; mixture was plated on LB+AMP and left to grow O/N


==Entry title==
==Team Allergy==
* Insert your content here.
* Gel extracte V9/V10 backbone
[[Image:V9v10.jpg|240px]]
''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:
[[Image:AmiRNA-insert-diagnostic.jpg|240px]]
* Digested Bet,LTP inserts with X+P, B21 with X+P+phosphatase
* Ligated, transformed
** 0-7: Bet, corresponding to 65..55 degrees C for stitching Tm (even spacing)
** 8-15: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing




Revision as of 14:54, 7 September 2010

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Team Fence

Colonies from yesterday's ligations

Gel extraction of EcR-Gal Bam Nco

  • applied 3x gel volume of QG buffer (in μL) to each respective tube
  • placed in 42°C bath until the gel was melted
  • added 1 gel volume of isopropanol to each, and vortexed
  • applied contents of each tube to a QIAquick spin collumn
  • spun for 1 min
  • added 500μL QG buffer (to dissolve any remaining agarose)
  • spun for 1 min
  • added 750μL PE buffer
  • spun for 1 min, discared flow-through
  • spun for another minute
  • placed QIAquick collumn in a fresh, labeled eppendorf tube, applied 50μL EB buffer to elute, and spun for 1 min


Nanodrop of Gal4 and EcR Gel Extractions

Plasmid Quantity (ng/μL) 260/280
Gal-EcR (1) 3.1 2.45
Gal-EcR (2) 4.5 1.72
Gal-EcR (3) 3.0 11.56
Gal-EcR (4) 3.6 2.21

Digestions

VP16

  • 1μL xba
  • 1μL Pst
  • 5μL VP16(2) miniprep
  • 1μL 10x FD green buffer
  • 2μL DH2O

5xGalUAS

  • 1μL xba
  • 1μL Pst
  • 2μL 5xGal4UAS (from Karmella)
  • 1μL 10x FD green buffer
  • 5μL DH2O

PhyB

  • 1μL Bam
  • 1μL Nco
  • 16μL PhyB
  • 2μL 10x FD green buffer

Sending EcR-Gal for sequencing

  • 5μL of each primer, either fwd or rev, at 1/20 dilution
  • 10μL of sample and DH2O, such that sample is at 500ng total mass

Sealed tubes with parafilm. Submited Gal-EcR 1, 7, 10, and 12 (the one's with inserts on the digestion gel from 8/4)

Team Flavor

Transformation of J45004

  • 5μL of registry DNA was transformed with 15μL TURBO cells; mixture was plated on LB+AMP and left to grow O/N

Team Allergy

  • Gel extracte V9/V10 backbone

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:

  • Digested Bet,LTP inserts with X+P, B21 with X+P+phosphatase
  • Ligated, transformed
    • 0-7: Bet, corresponding to 65..55 degrees C for stitching Tm (even spacing)
    • 8-15: LTP, corresponding to 65.55 degrees C during stitching annealing, even spacing



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