Team:Cornell/Week 10

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Revision as of 01:51, 10 August 2011

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.

Monday, August 8

Morning lab work done by: Youjin Cho and James Mathew

Objective

• Transform the overnight ligation of VioA/VioB+Avi-tag+pZE12.
• Send the miniprepped samples (GFP, RFP, VioE) from Saturday.
• Miniprepped the additional VioE samples that was grown overnight.

Transformation

• Desalted the ligation reaction samples(VioA/VioB+Avitag+pZE12) on a membrane for 15minutes.
• After adding the samples into DH5α cell lines, electroporated them and put into shaker for 45minutes.
• The samples were plated on the plate with ampicilin.

Sequencing

• Using the reverse primer dilution, the samples were sent to sequencing.

GFP+Avitag+pZE12 - 1

GFP+Avitag+pZE12 - 2

GFP+Avitag+pZE12 - 3

RFP+Avitag+pZE12 - 1

RFP+Avitag+pZE12 - 2

RFP+Avitag+pZE12 - 3

VioE+Avitag+pZE12 - 2

Miniprepping the VioE samples

• The samples were miniprepped using standard Qiagen Miniprep protocol.

VioE+Avitag+pZE12 - 4

VioE+Avitag+pZE12 - 5

VioE+Avitag+pZE12 - 6

Evening lab work done by: Charlie Chung

• Checked to see if colonies were ready to pick from transformed plates of (vioA + Avi-Tagged pZE12 backbone) and (vioB + Avi-Tagged pZE12 backbone).

• vioA plate is showing early signs of a good number of colonies. Too small for picking, however.
• vioB and Control plates look pretty empty (but just may not have grown up enough to tell).

• Will be best if colonies were picked Tuesday (tomorrow) morning. Please find the 3 plates still in the 37°C incubator.

Tuesday, August 9

Evening lab work done by: Youjin Cho

• Picked colonies from transformed plates of (vioA + Avi-Tagged pZE12 backbone) and (vioB + Avi-Tagged pZE12 backbone.

VioA 1,2,3

VioB 1,2,3

Wednesday, August 10

Thursday, August 11

Friday, August 12

Saturday, August 13