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Team:Cornell/Week 10
From 2011.igem.org
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==Wednesday== | ==Wednesday== | ||
Revision as of 19:42, 8 August 2011
Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
August 7th - August 13th
Sunday, August 7
Morning lab work done by: Youjin Cho and Claire Paduano
- Objective
- Prepare VioE in Avi-Tagged pZE12 vector backbone for VioA and VioB gene inserts
- Digestion Setup
- 23.65μL H2O
- 18.6μL VioE in Avi-Tagged pZE12 vector backbone (1μg)
- 5μL 10x NEBuffer 2
- 0.5μL 100x BSA
- 1.25μL KpnI
- 1μL HindIII
- 50μL Total
- KpnI has only 75% efficiency in Buffer 2, so added 0.25uL more KpnI to digestion mixture
- Incubate in 37°C water bath for 2 hours
Afternoon lab work done by: Jim Mathew, Charlie Chung
- Objective
- Ligate vioA and vioB genes onto their own pZE12 vector backbone, in place of the vioE that is now digested out
- Ligation Reaction Setup
- vioA + Avi-Tagged pZE12 vector backbone
- 11.4μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
- 3.3μL vioA gene insert (cut with KpnI & HindIII)
- 2.3μL H2O
- 2μL 10x T4 DNA ligase buffer
- 1μL T4 DNA ligase
- 20μL Total
- vioB + Avi-Tagged pZE12 vector backbone
- 9μL vioB gene insert (cut with KpnI & HindIII)
- 8μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
- 2μL 10x T4 DNA ligase buffer
- 1μL T4 DNA ligase
- 20μL Total
- Control Ligation for vioA and vioB Gene Inserts
- 11.4μL pZE12 backbone (cut with KpnI & HindIII; former vioE gene digested out)
- 5.6μL H2O (gene insert volumes go toward water volume)
- 2μL 10x T4 DNA ligase buffer
- 1μL T4 DNA ligase
- 20μL Total
- Incubate all ligation reaction constructs overnight in 16°C waterbath.
Evening lab work done by: Charlie Chung
- Objective
- Because previous DH5α colonies transformed with (vioE + Avi-Tagged backbone) are very small, in addition to one failed 5mL culture and one discarded Miniprep sample, incubated plate for four more hours.
- Picked three new colonies. Labeled as "vioE + Avi + BB #4, 5, 6"
- 5mL LB cultures with 5μL ampicillin incubated overnight in 37°C shaker of Room 304.
