IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/08: Difference between revisions
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*Transformed two pDUET expression vectors for Miniprep | *Transformed two pDUET expression vectors for Miniprep | ||
===Ligated Miraculin/Brazzein to a StrepII tag=== | |||
{| border="1" | |||
|+ ''Ligation Reactions'' | |||
! !! Miraculin xbaI/pstI w/ B15 speI/pstI!! Miraculin ecoRI/speI w/ B15 ecoRI/xbaI!! Brazzein xbaI/pstI w/ B15 speI/pstI !! Brazzein ecoRI/speI w/ B15 ecoRI/xbaI!! Control B15 speI/pstI!! Control B15 ecoRI/xbaI | |||
|- | |||
! DNA Insert | |||
| align="center" | 3 || align="center" | 4|| align="center" | 4|| align="center" | 2|| align="center" | 0||align="center"| 0 | |||
|- | |||
! T4 DNA ligase buffer (10x) | |||
| align="center" | 2 || align="center" | 2 || align="center" | 2|| align="center" | 2|| align="center" | 2|| align="center" |2 | |||
|- | |||
! diH<sub>2</sub>O | |||
| align="center" | 11 || align="center" | 10 || align="center" | 10|| align="center" | 12|| align="center" | 14|| align="center"| 14 | |||
|- | |||
! T4 DNA ligase | |||
| align="center" | 1 || align="center" | 1 || align="center" | 1|| align="center" | 1|| align="center" | 1|| align = "center"| 1 | |||
|- | |||
! DNA Backbone | |||
| align="center" | 3 || align="center" | 3|| align="center" | 3|| align="center" | 3|| align="center" | 3|| align="center" |3 | |||
|- | |||
|} | |||
* We then transformed the ligated plasmids into Turbo e. coli and plated the bacteria on Amp plates. Plates were left in the 37°C incubator overnight. | |||
==Team Fence== | ==Team Fence== |
Revision as of 07:48, 9 July 2010
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Team Flavor
Ligated Miraculin/Brazzein to a StrepII tag
Team Fence
Gel extraction was performed according to the QIAgen gel extraction kit protocol using a microcentrifuge. Team AllergyamiRNA
Allergen Panel
LTP 1,2,3: 265.5 ng/uL, 507.1 ng/uL, 275.5 ng/uL; GerA 1,2,3,4: 283.1 ng/uL, 185.9 ng/uL, 182.4 ng/uL, 314.7 ng/uL; Bet 2A 1,2,3,4: 246.6 ng/uL, 251.4ng/uL, 550.3 ng/uL, 235.8 ng/uL; Bet 2S 1,2,3,4,5,6: 613 ng/uL, 296.8 ng/uL, 333.1 ng/uL, 467.3 ng/uL, 230.3 ng/uL, 129.4 ng/uL; Bet 1A 1,2,3,4,5: 505.8 ng/uL, 632.1 ng/uL, 561.7 ng/uL, 594.4 ng/uL, 609.2 ng/uL
[[Image: |240px]] PDK intron
Gel of gradient PCR of the 741bp PDK intron out of pHANNIBAL and pKANNIBAL. Lanes are (left to right, temperatures are annealing temperatures used in the gradient PCR) 1kb+ ladder, pHAN 50ºC, pKAN 50ºC, pHAN 55ºC, pKAN 55ºC, pHAN 60ºC, pKAN 60ºC, pHAN 65ºC, pKAN 65ºC. It appears that pHANNIBAL worked at 50, 55ºC but pKANNIBAL didn't. Concentration: hannibal pdk 12.7 ng/uL kannibal pdk: 15.6 ng/uL Did a PCR of the PCR product Annealing Temps: hannibal pdk (50 C); kannibal pdk (55 C) Extension Time: 30 sec 12 reactions (6 hannibal pdk; 6 kannibal pdk)
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