IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/04: Difference between revisions

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[[Image:EcR-Gal_lig_VP16.4_Pcr_8-04.jpg]]
[[Image:EcR-Gal_lig_VP16.4_Pcr_8-04.jpg]]


===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
===Ligated EcR to Gal4 and Barnase to Strep===
Sealed tubes with parafilm.
Did not submit Gal-EcR 1,4, or 9 on account of insufficient DNA to both sequence and use it.


==Team Flavor==
==Team Flavor==

Revision as of 14:54, 7 September 2010

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Team Allergy

  • amiRNA fragment production appears to have worked at every Tm we tried (click images for details):

  • V9/V10
    • Diagnostic digest of our gel :

[[Image: |240 px]]

(Should see ~ 7kb band and an 840 band)

    • Realized that we hadn't gel purified our inserts
    • Gel purification of inserts (entire ihpRNA parts)

Gel 1 [[Image: |240px|Gel 2]] Gel 1: Ladder, 9, 11c1, 11c2, ladder, 25c1, 28c1, 28c2, 36c1, 36c2, ladder Gel 2: Ladder, 37c1, 37 c2, 38c1, 38c2, ladder

Team Fence

PCR of VP16.3

60μL reaction:

  • 1.5 μL pfx polymerase
  • 3μL DNTPs
  • 18μL enhancer buffer
  • 1.5μL MgSO4
  • 3μL 1/10 dilution VP16.Hind.Fwd
  • 3μL 1/10 dilution VP16.Rev
  • 3μL 1/10 dilution of VP16 (1)
  • 12μL amp buffer
  • 5μL DH2O

Using program: 

  1=94°C for 2:00
  2=94°C for :15
  3=60/68°C for :30
  4=68°C for :10
  5=Goto 2, 30 times
  6=4.0°C forever
  7=end

Digestion of Gal-EcR

BamH1 and Nco1 12 digestions, one for each Gal-EcR miniprep Each:

  • 1μL BamH1
  • 1μL Nco1
  • 1μL 10x FD green buffer
  • 2-7μL EcR-Gal miniprep
  • 5-0μL DH2O, to bring total volume to 10μL

Ran a 1% gel of the digest

1 2 3 4 5 6 7 8 9 10 11 12 13 14
EcR Digest 1 EcR Digest 2 EcR Digest 3 EcR Digest 5 EcR Digest 6 EcR Digest 7 EcR Digest 8 EcR Digest 9 EcR Digest 10 EcR Digest 11 EcR Digest 12 KB+ ladder


Ligated EcR to Gal4 and Barnase to Strep

Team Flavor

Confirmation Digests

  • Ran confirmation digests of miniprepped J45004-1, J45004-2, J45017-1, J45017-2
    • Digested approximately 500ng with xbaI/speI
  • Ran confirmation digests of miniprepped Miraculin N strep+stop and V24, Miraculin C strep+stop and V24, Brazzein N strep+stop and V24, Brazzein C strep+stop and V24.
    • Digested 400-500ng with notI/speI

Retry Ligating j45004 and v0120

  • Used PCR purified J45004 that had been previously digested with xbaI/pstI
  • Used v0120 from Team Vector that they had digested with xbaI/pstI and used in a successful ligation
  • 3:1 insert to backbone ration used in ligation
    • 52ng of insert and 50ng v0120 backbone
    • J45004 insert is 1100 bp
  • Transformed and plated on LB+Amp plates and left overnight


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