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

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==Team Allergy==
==Team Allergy==
*amiRNA
*amiRNA fragment production appears to have worked at every Tm we tried (click images for details):
[[Image:AmiRNA-try-3.jpg|240px]][[Image:AmiRNA_pcr_8.3|240px]]
 
*V9/V10  
*V9/V10  
**Diagnostic digest of our gel :
**Diagnostic digest of our gel :
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Gel 2: Ladder, 37c1, 37 c2, 38c1, 38c2, 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 MgSO<sub>4</sub>
*3μL 1/10 dilution VP16.Hind.Fwd
*3μL 1/10 dilution VP16.Rev
*3μL 1/10 dilution of VP16 (1)
*12μL amp buffer
*5μL DH<sub>2</sub>O
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 DH<sub>2</sub>O, to bring total volume to 10μL
Ran a 1% gel of the digest
{| class="wikitable" border ="1px solid black"
|-
|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
|-
|}
[[Image:EcR-Gal_lig_VP16.4_Pcr_8-04.jpg]]
===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


[[Image:J45004_017_mira_brazz_ss_annotated.jpg||350px]]


===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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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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