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From 2011.igem.org

August 21st - August 27th

Sunday, August 21

Evening lab work done by: Youjin Cho, Charlie Chung

Picking Colonies and Starting Up Cultures of VioB Ligation Reaction

• (AviTagged pZE12 backbone) control plate shows no growth of colonies
• (VioB + AviTagged pZE12 backbone) plate shows numerous colonies

- Picked three colonies (VioB + AviTag + BB #1, 2, 3) and started 3mL cultures in LB with 3μL ampicillin

- Incubated overnight in the 37°C shaker of Room 304

PCR Reaction Setup of GFP (because sequencing results were negative)

38.5μL ddH2O

5μL 10x buffer

2.5μL dNTP

1μL forward GFP primer

1μL reverse GFP primer

1μL (pZE12+GFP) backbone template

1μL Vent DNA polymerase

50μL Total

• Annealing temperature of 54.2°C
• 1 minute extension cycle

Monday, August 22

Morning lab work done by: Youjin Cho

Objective

• Miniprep the VioB + AviTag + pZE12 samples and submit for sequencing

Miniprep and Sequencing

• Miniprepped the VioB + AviTag + pZE12 (1,2,3) samples using standard Qiagen Miniprep protocol
• Using 1μL of pZE12 reverse primer dilution and 17μL of each template, prepared and submitted the samples for sequencing.

Order number: 10255430

Tuesday, August 23

Afternoon lab work done by: Claire Paduano, Maneesh Gupta, Youjin Cho, Charlie Chung

Objective

• Set up new ligation of GFP and AviTagged backbone
• Send out gel purified GFP PCR reaction product to troubleshoot why our procedure isn't working

• Conducted gel electrophoresis on PCR reaction product of GFP

- Visualization of migrated DNA fragments does not show a GFP band around 750bp

• Redid PCR on GFP

- Note: This time, we made sure to add 1µL MgSO4

37.5μL ddH2O

5μL 10x buffer

1μL 25mM dNTP

1μL MgSO4

1μL forward GFP primer

1μL reverse GFP primer

1μL (pZE12+GFP) backbone template

1μL Vent DNA polymerase

48.5μL Total

• Annealing temperature of 54.2°C
• 1 minute extension cycle

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Gel electrophoresis

- Visualization shows GFP band around 750bp

Gel purified GFP product

Digestion Setups

• GFP insert

36μL H2O

6.5μL GFP (1μg)

5μL 10x NEBuffer 4

0.5μL 100x BSA

1.0μL KpnI-HF

1μL SphI-HF

50μL Total

• AviTagged pZE12 vector backbone

32.9μL H2O

9.6μL VioE in AviTagged pZE12 vector backbone (1μg)

5μL 10x NEBuffer 4

0.5μL 100x BSA

1.0μL KpnI-HF

1μL SphI-HF

50μL Total

Incubate in 37°C water bath for 2 hours

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Evening lab work done by: James Mathew, Maneesh Gupta, Claire Paduano

• Gel purified digested Avi-Tagged Backbone

Ligation

• GFP + Avi-Tagged Backbone

5.8μL Avi-Tagged pZE12 vector backbone

7.97μL GFP

3.4μL H2O

2.0μL T4 DNA Ligase Buffer

1.0μL T4 DNA Ligase

20μL Total

In above reaction, volume of plasmid vector added corresponds with 50ng backbone.

Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs.

• Same volume of vector backbone, buffer, ligase
• Volumes of all inserts (i.e. gene) go toward volume of H2O

After ligation setup, incubate at 16degC overnight.

Wednesday, August 24

Thursday, August 25

Lab work done by: Youjin Cho

Transformation for GFP

• Desalted the ligation of GFP into AviTag + pZE12 backbone and the control on a membrane for 15 minutes
• Transformed the ligation GFP sample and control in DH5α electrocomptent cells via electroporation
• After shaking the cells in 37°C shaker for 50 minutes, plated onto agar treated with ampicilin and incubated overnight at 37°C

Friday, August 26

Morning lab work done by: Youjin Cho

• Checked the plates for GFP+Avitag+pZE12, but the ratio of ligation to control was bad.

Afternoon lab work done by: James Mathew, Maneesh Gupta, Claire Paduano

• Set up ligation for a second time since previous ligation was bad

Ligation

• GFP + AviTagged Backbone

3.52μL AviTagged pZE12 vector backbone

7.97μL GFP

5.54μL H2O

2.0μL T4 DNA ligase buffer

1.0μL T4 DNA ligase

20μL Total

In above reaction, volume of plasmid vector added corresponds with 50ng backbone

Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs

• Same volume of vector backbone, buffer, ligase
• Volumes of all inserts (i.e. gene) go toward volume of H2O

After ligation setup, incubate at 16°C overnight

Saturday, August 27

Weill Hall lab work done by: Claire Paduano, James Mathew

Objective

• Coat microfluidic device with steptavidin.
• Run biotinylated fluorescent ATTO dye to test binding http://www.sigmaaldrich.com/img/assets/12980/atto_flyer.pdf ATTO dye info

Streptavidin coating and fluorescent labeling

• Protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046

1) 45 min in 4% (by volume) MPTMS in ethanol

2) 20 min in 1mM GMBS

3) 45 min in 25ng/mL NeutrAvidin in PBS

4) 20 min (at flow rate 5ul/min) 1mM ATTO-590 dye

• Took photos of channel (bright field), negative control (streptavidin coated, no fluorescent probe), and streptavidin coated channel with fluorescent probe flushed through: