Team:Cornell/Week 9
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- | {{:Team:Cornell/Templates/ | + | {{:Team:Cornell/Templates/Menu}} |
{{:Team:Cornell/Templates/hideHeader}} | {{:Team:Cornell/Templates/hideHeader}} | ||
{{:Team:Cornell/Templates/MediaMenu}} | {{:Team:Cornell/Templates/MediaMenu}} | ||
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==Monday, August 1== | ==Monday, August 1== | ||
Lab work done by: Jim Mathew | Lab work done by: Jim Mathew | ||
- | :*Sent | + | :*Sent VioE and AviTag + backbone in for sequencing |
==Tuesday, August 2== | ==Tuesday, August 2== | ||
Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo | Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo | ||
- | |||
:'''Microfluidics''' | :'''Microfluidics''' | ||
- | ::*Dr. Archer showed us how to use the clean room | + | ::*Dr. Archer showed us how to use the clean room |
- | ::*Made a SU-8 master of our device | + | ::*Made a SU-8 master of our device |
::*Poured PDMS and baked overnight at 60°C | ::*Poured PDMS and baked overnight at 60°C | ||
- | + | ::*Followed protocol that is used by Biomedical Engineering lab class and is provided by Dr. Archer | |
- | :: | + | |
==Wednesday, August 3== | ==Wednesday, August 3== | ||
Line 27: | Line 25: | ||
:'''Microfluidics''' | :'''Microfluidics''' | ||
- | ::*Dr. Archer showed us how to cut out PDMS devices and seal to clean glass slides using plasma cleaner | + | ::*Dr. Archer showed us how to cut out PDMS devices and seal to clean glass slides using plasma cleaner |
::*Tested chips for blockages in flow | ::*Tested chips for blockages in flow | ||
- | :::Three working devices, one with complete blockage in the channel | + | :::- Three working devices, one with complete blockage in the channel |
- | + | ||
::*Poured more PDMS and baked overnight at 60°C | ::*Poured more PDMS and baked overnight at 60°C | ||
- | :: | + | ::*Followed protocol used in Biomedical Engineering lab class and provided by Dr. Archer |
==Thursday, August 4== | ==Thursday, August 4== | ||
Line 38: | Line 35: | ||
:'''Objective''' | :'''Objective''' | ||
- | ::Prepare Avi-Tagged pZE12 vector backbone for GFP, RFP, and | + | ::Prepare Avi-Tagged pZE12 vector backbone for GFP, RFP, and VioE gene inserts |
:'''Digestion Setup in Duplicate''' | :'''Digestion Setup in Duplicate''' | ||
:::32.6μL H2O | :::32.6μL H2O | ||
- | :::9.9μL | + | :::9.9μL AviTagged pZE12 vector backbone (2μg) |
:::5μL 10x NEBuffer 4 | :::5μL 10x NEBuffer 4 | ||
:::0.5μL 100x BSA | :::0.5μL 100x BSA | ||
Line 53: | Line 50: | ||
::#Run vector backbone through agarose gel electrophoresis | ::#Run vector backbone through agarose gel electrophoresis | ||
:'''Gel Extraction and Purification of Digested Backbone''' | :'''Gel Extraction and Purification of Digested Backbone''' | ||
- | ::*Followed standard Qiagen Gel Extraction protocol for pZE12 vector backbone that is now | + | ::*Followed standard Qiagen Gel Extraction protocol for pZE12 vector backbone that is now AviTagged and digested with KpnI and SphI |
::*NanoDrop spectrophotometry on the duplicate samples reported 12.7ng/μL and 14.2ng/μL | ::*NanoDrop spectrophotometry on the duplicate samples reported 12.7ng/μL and 14.2ng/μL | ||
- | |||
---- | ---- | ||
- | |||
Afternoon lab work done by: Claire Paduano, Charlie Chung | Afternoon lab work done by: Claire Paduano, Charlie Chung | ||
- | |||
:'''Objective''' | :'''Objective''' | ||
- | ::Ligation of GFP, RFP, and | + | ::Ligation of GFP, RFP, and VioE genes with AviTagged pZE12 backbone |
:'''Ligation''' | :'''Ligation''' | ||
- | ::*''GFP + | + | ::*''GFP + AviTagged Backbone'' |
- | ::::10.6μL | + | ::::10.6μL AviTagged pZE12 vector backbone |
::::3.6μL GFP | ::::3.6μL GFP | ||
::::2.8μL H2O | ::::2.8μL H2O | ||
- | ::::2.0μL T4 DNA | + | ::::2.0μL T4 DNA ligase buffer |
- | ::::<u>1.0μL T4 DNA | + | ::::<u>1.0μL T4 DNA ligase</u> |
::::20μL Total | ::::20μL Total | ||
- | ::*''RFP + | + | ::*''RFP + AviTagged Backbone'' |
- | ::::10.3μL | + | ::::10.3μL AviTagged pZE12 vector backbone |
::::6.7μL RFP | ::::6.7μL RFP | ||
- | ::::2.0μL T4 DNA | + | ::::2.0μL T4 DNA ligase buffer |
- | ::::<u>1.0μL T4 DNA | + | ::::<u>1.0μL T4 DNA ligase</u> |
::::20μL Total | ::::20μL Total | ||
- | ::*'' | + | ::*''VioE + AviTagged Backbone'' |
- | ::::10.6μL | + | ::::10.6μL AviTagged pZE12 vector backbone |
- | ::::4.1μL | + | ::::4.1μL VioE |
::::2.3μL H2O | ::::2.3μL H2O | ||
- | ::::2.0μL T4 DNA | + | ::::2.0μL T4 DNA ligase buffer |
- | ::::<u>1.0μL T4 DNA | + | ::::<u>1.0μL T4 DNA ligase</u> |
::::20μL Total | ::::20μL Total | ||
- | ::In all above reactions, volume of plasmid vector added corresponds with 100ng backbone | + | ::In all above reactions, volume of plasmid vector added corresponds with 100ng backbone |
- | ::Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs | + | ::Prepare control ligation reactions (inserts replaced with H2O) for each of the above constructs |
:::*Same volume of vector backbone, buffer, ligase | :::*Same volume of vector backbone, buffer, ligase | ||
:::*Volumes of all inserts (i.e. gene) go toward volume of H2O | :::*Volumes of all inserts (i.e. gene) go toward volume of H2O | ||
- | ::After ligation setup, incubate in 16°C | + | ::After ligation setup, incubate in 16°C water bath overnight |
- | + | ::Ligation reaction volumes were calculated using a formulated spreadsheet (see ''Protocol'' page) that is based on 100ng of vector backbone and a 3:1 molar ratio of insert:vector | |
- | ::Ligation reaction volumes were calculated using a formulated spreadsheet (see ''Protocol'' page | + | |
:'''Reference for Inserts and Vector Backbone''' | :'''Reference for Inserts and Vector Backbone''' | ||
::RFP gene - 678bp | ::RFP gene - 678bp | ||
Line 97: | Line 90: | ||
==Friday, August 5== | ==Friday, August 5== | ||
- | Lab work done by: Youjin Cho | + | Lab work done by: Youjin Cho, Claire Paduano |
- | + | ||
:'''Objective''' | :'''Objective''' | ||
- | ::Transform ligation of GFP/RFP/ | + | ::Transform ligation of GFP/RFP/VioE + AviTagged backbone |
- | + | ||
:#Desalted overnight ligation | :#Desalted overnight ligation | ||
:#Transformed the samples into DH5α electrocompetent cells | :#Transformed the samples into DH5α electrocompetent cells | ||
- | |||
- | |||
- | |||
:'''Group Meeting''' | :'''Group Meeting''' | ||
- | ::*Just finished transforming GFP/RFP/ | + | ::*Just finished transforming GFP/RFP/VioE + AviTag + pZE12 backbone |
- | ::*If the | + | ::*If the VioE gene successfully inserts and ligates with the AviTagged backbone, then we can ligate in VioA and VioB this weekend |
- | ::*Goal: to treat PDMS devices to coat with streptavidin and bind | + | ::*Goal: to treat PDMS devices to coat with streptavidin and bind AviTagged GFP and RFP to device |
==Saturday, August 6== | ==Saturday, August 6== | ||
Lab work done by: Youjin Cho, Charlie Chung | Lab work done by: Youjin Cho, Charlie Chung | ||
- | *Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol) | + | :*Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol) |
- | :NOTE: White masses were floating in bacteria culture -- potential contamination? | + | ::<u>NOTE</u>: White masses were floating in bacteria culture -- potential contamination? |
- | *NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product | + | :*NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product |
- | ::RFP (Colony 1) + | + | ::RFP (Colony 1) + AviTagged pZE12 vector backbone = 53.2ng/µL |
- | ::RFP (Colony 2) + | + | ::RFP (Colony 2) + AviTagged pZE12 vector backbone = 63.4ng/µL |
- | ::RFP (Colony 3) + | + | ::RFP (Colony 3) + AviTagged pZE12 vector backbone = 76.6ng/µL |
- | ::GFP (Colony 1) + | + | ::GFP (Colony 1) + AviTagged pZE12 vector backbone = 65.7ng/µL |
- | ::GFP (Colony 2) + | + | ::GFP (Colony 2) + AviTagged pZE12 vector backbone = 65.5ng/µL |
- | ::GFP (Colony 3) + | + | ::GFP (Colony 3) + AviTagged pZE12 vector backbone = 46.6ng/µL |
- | :: | + | ::VioE (Colony 1) did not grow in its culture tube |
- | :: | + | ::VioE (Colony 2) + AviTagged pZE12 vector backbone = 53.8ng/µL |
- | ::DNA from | + | ::DNA from VioE (Colony 3) was accidentally discarded...Charlie is extremely ashamed. |
Latest revision as of 17:23, 18 September 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 |
July 31st - August 6th
Sunday, July 31
Monday, August 1
Lab work done by: Jim Mathew
- Sent VioE and AviTag + backbone in for sequencing
Tuesday, August 2
Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo
- Microfluidics
- Dr. Archer showed us how to use the clean room
- Made a SU-8 master of our device
- Poured PDMS and baked overnight at 60°C
- Followed protocol that is used by Biomedical Engineering lab class and is provided by Dr. Archer
Wednesday, August 3
Microfluidics work done by: Jim Mathew, Nick Kramer, Dan Levine, Claire Paduano, Youjin Cho, Bill Jo
- Microfluidics
- Dr. Archer showed us how to cut out PDMS devices and seal to clean glass slides using plasma cleaner
- Tested chips for blockages in flow
- - Three working devices, one with complete blockage in the channel
- Poured more PDMS and baked overnight at 60°C
- Followed protocol used in Biomedical Engineering lab class and provided by Dr. Archer
Thursday, August 4
Morning lab work done by: Jim Mathew
- Objective
- Prepare Avi-Tagged pZE12 vector backbone for GFP, RFP, and VioE gene inserts
- Digestion Setup in Duplicate
- 32.6μL H2O
- 9.9μL AviTagged pZE12 vector backbone (2μg)
- 5μL 10x NEBuffer 4
- 0.5μL 100x BSA
- 1μL KpnI-HF (HF = high fidelity)
- 1μL SphI-HF
- 50μL Total
- Incubate in 37°C water bath for 2 hours
- Dephosphorylation of 5' Ends of Vector Backbone
- Add 1μL of Calf Intestine Alkaline Phosphatase (CIAP) to digested vector backbone in order to prevent self-ligation without gene insert included
- Incubate at 50°C for 5 minutes
- Run vector backbone through agarose gel electrophoresis
- Gel Extraction and Purification of Digested Backbone
- Followed standard Qiagen Gel Extraction protocol for pZE12 vector backbone that is now AviTagged and digested with KpnI and SphI
- NanoDrop spectrophotometry on the duplicate samples reported 12.7ng/μL and 14.2ng/μL
Afternoon lab work done by: Claire Paduano, Charlie Chung
- Objective
- Ligation of GFP, RFP, and VioE genes with AviTagged pZE12 backbone
- Ligation
- GFP + AviTagged Backbone
- 10.6μL AviTagged pZE12 vector backbone
- 3.6μL GFP
- 2.8μL H2O
- 2.0μL T4 DNA ligase buffer
- 1.0μL T4 DNA ligase
- 20μL Total
- RFP + AviTagged Backbone
- 10.3μL AviTagged pZE12 vector backbone
- 6.7μL RFP
- 2.0μL T4 DNA ligase buffer
- 1.0μL T4 DNA ligase
- 20μL Total
- VioE + AviTagged Backbone
- 10.6μL AviTagged pZE12 vector backbone
- 4.1μL VioE
- 2.3μL H2O
- 2.0μL T4 DNA ligase buffer
- 1.0μL T4 DNA ligase
- 20μL Total
- In all above reactions, volume of plasmid vector added corresponds with 100ng 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 in 16°C water bath overnight
- Ligation reaction volumes were calculated using a formulated spreadsheet (see Protocol page) that is based on 100ng of vector backbone and a 3:1 molar ratio of insert:vector
- Reference for Inserts and Vector Backbone
- RFP gene - 678bp
- GFP gene - 717bp
- vioE gene - 576bp
- pZE12 vector - 2340bp
Friday, August 5
Lab work done by: Youjin Cho, Claire Paduano
- Objective
- Transform ligation of GFP/RFP/VioE + AviTagged backbone
- Desalted overnight ligation
- Transformed the samples into DH5α electrocompetent cells
- Group Meeting
- Just finished transforming GFP/RFP/VioE + AviTag + pZE12 backbone
- If the VioE gene successfully inserts and ligates with the AviTagged backbone, then we can ligate in VioA and VioB this weekend
- Goal: to treat PDMS devices to coat with streptavidin and bind AviTagged GFP and RFP to device
Saturday, August 6
Lab work done by: Youjin Cho, Charlie Chung
- Miniprepped 5mL cultures of transformed DH5α electrocompetent bacteria (standard Qiagen Miniprep protocol)
- NOTE: White masses were floating in bacteria culture -- potential contamination?
- NanoDrop spectrophotometry to determine DNA concentration of 50µL elution product
- RFP (Colony 1) + AviTagged pZE12 vector backbone = 53.2ng/µL
- RFP (Colony 2) + AviTagged pZE12 vector backbone = 63.4ng/µL
- RFP (Colony 3) + AviTagged pZE12 vector backbone = 76.6ng/µL
- GFP (Colony 1) + AviTagged pZE12 vector backbone = 65.7ng/µL
- GFP (Colony 2) + AviTagged pZE12 vector backbone = 65.5ng/µL
- GFP (Colony 3) + AviTagged pZE12 vector backbone = 46.6ng/µL
- VioE (Colony 1) did not grow in its culture tube
- VioE (Colony 2) + AviTagged pZE12 vector backbone = 53.8ng/µL
- DNA from VioE (Colony 3) was accidentally discarded...Charlie is extremely ashamed.