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" | |||
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|9 | |||
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|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]] | [[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
[[Image: |240 px]] (Should see ~ 7kb band and an 840 band)
[[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 FencePCR of VP16.360μL reaction:
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-EcRBamH1 and Nco1 12 digestions, one for each Gal-EcR miniprep Each:
Ran a 1% gel of the digest
Ligated EcR to Gal4 and Barnase to StrepTeam FlavorConfirmation Digests
Retry Ligating j45004 and v0120
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