Team:Cornell/Week 7

From 2011.igem.org

(Difference between revisions)

Latest revision as of 17:23, 18 September 2011

Results | Protocol | Notebook | Parts Submitted

July 17th - July 23rd

Sunday, July 17

Lab work done by: Alyssa Henning, Bill Jo

Miniprepped 1 sample of GFP + AviTag (the second sample got messed up)
Ran a gel on the RFP that was amplified via PCR on Saturday
Gel purification and extraction
Sean will inoculate 6 tubes of GFP-containing bacteria -- 1 tube for us, 5 tubes for CURIE (Cornell iGEM's outreach program)

Monday, July 18

Lab work done by: Charlie Chung, Youjin Cho

Gel purified 2 RFP PCR samples from Friday
Miniprep 2 samples of GFP + AviTag

Tuesday, July 19

Lab work done by: James Mathew

Submitted GFP + AviTag for synthesis
Submitted pZE12 backbone for subcloning of light sensor

Wednesday, July 20

Lab work done by: James Mathew

Set up ligation reaction of pZE-12 backbone with the primer dimer insert

1. Digestion of Backbone

- 26µl Backbone plasmid = 2µg DNA

- 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF)

- 1µl SphI & 1µl ClaI

- 16.5µl H2O

left in water bath for one hour

- 1µl CIAP added to digestion reaction

left in water bath for 30 minutes

2. Ran Digestion Product through gel for 30 minutes at 120V

Lane 1: 2µl 1kb Ladder + 2µl 6x loading dye + 8µl H2O

Lane 2: 25µl digestion reaction + 5µl 6x loading dye

Lane 3: 25µl digestion reaction + 5µl 6x loading dye

3. Gel Purification using Qiagen Kit

4. NanoDrop of Samples

5. Ligation Reaction

Ligation done overnight for transformation on 7/21/11

Thursday, July 21

Lab work done by: James Mathew

Miniprepped Vio operon (Cambridge 2009)

Lab work done by: Youjin Cho, Charlie Chung, Alyssa Henning

Reconstituted VioA, VioB, and VioE forward and reverse primers
Set up PCR for VioA, VioB, and VioE
Aborted transformation of pZE12 plasmid + AviTag primer dimer because reverse primers for AviTag were incorrect
Redesigned reverse primers for AviTag to order on Friday

Friday, July 22

Gel electrophoresis

Lab work done by: James Mathew, Claire Paduano

PCR gel purification of VioA, VioB, and VioE

Saturday, July 23

Lab work done by: James Mathew, Claire Paduano

Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and AviTag primer dimer

@@ Line 1: / Line 1: @@
-{{:Team:Cornell/Templates/test}}
+{{:Team:Cornell/Templates/Menu}}
 {{:Team:Cornell/Templates/hideHeader}}
 {{:Team:Cornell/Templates/MediaMenu}}
@@ Line 15: / Line 15: @@
 ==Monday, July 18==
-Lab work done by: Charlie Chung & Youjin Cho
+Lab work done by: Charlie Chung, Youjin Cho
-*Gel purified 2 RFP PCR samples from Friday.
+:*Gel purified 2 RFP PCR samples from Friday
-*Miniprepped 2 samples of GFP + avitag.
+:*Miniprep 2 samples of GFP + AviTag
 ==Tuesday, July 19==
-Lab Work Done By: James Mathew
+Lab work done by: James Mathew
-*Submitted GFP+avitag genes for synthesis
+:*Submitted GFP + AviTag for synthesis
-*Submitted pZE-12 backbone for subcloning of light sensor
+:*Submitted pZE12 backbone for subcloning of light sensor
 ==Wednesday, July 20==
 Lab work done by: James Mathew
-*'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert.'''
+*'''Set up ligation reaction of pZE-12 backbone with the primer dimer insert'''
 ::1. <u>Digestion of Backbone</u>
-:::- 26 ul Backbone plasmid = 2 ug DNA
+:::- 26µl Backbone plasmid = 2µg DNA
-:::- 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
+:::- 5µl NEBuffer 4 (optimal for SphI-HF and ClaI-HF)
-:::- 1 ul SphI & 1 ul ClaI
+:::- 1µl SphI & 1µl ClaI
-:::- 16.5 ul H20
+:::- 16.5µl H2O
 ::::*left in water bath for one hour
-:::- 1 ul CIAP added to digestion reaction
+:::- 1µl CIAP added to digestion reaction
 ::::*left in water bath for 30 minutes
+::2. <u>Ran Digestion Product through gel for 30 minutes at 120V</u>
-::2. <u>Ran Digestion Product through gel for 30 minutes at 120 V.</u>
+::::Lane 1: 2µl 1kb Ladder + 2µl 6x loading dye + 8µl H2O
-::::Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20
+::::Lane 2: 25µl digestion reaction + 5µl 6x loading dye
-::::Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
+::::Lane 3: 25µl digestion reaction + 5µl 6x loading dye
-::::Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
 ::3. <u>Gel Purification using Qiagen Kit</u>
 ::4. <u>NanoDrop of Samples</u>
-::::Concentration:
-::::Label:
 ::5. <u>Ligation Reaction</u>
-::::Label:
+:*Ligation done overnight for transformation on 7/21/11
-*Ligation done overnight for transformation on 7/21/11
 ==Thursday, July 21==
 Lab work done by: James Mathew
-*miniprepped Vio operon (Cambridge 2009)
+*Miniprepped Vio operon (Cambridge 2009)
+Lab work done by: Youjin Cho, Charlie Chung, Alyssa Henning
-Lab work done by: Youjin Cho, Charlie Chung, and Alyssa Henning
 *Reconstituted VioA, VioB, and VioE forward and reverse primers
 *Set up PCR for VioA, VioB, and VioE
-*Aborted transformation of pZE-12 plasmid + avitag primer dimer because reverse primers for avitag were incorrect.
+*Aborted transformation of pZE12 plasmid + AviTag primer dimer because reverse primers for AviTag were incorrect
-*Redesigned reverse primers for avitag to order on Friday
+*Redesigned reverse primers for AviTag to order on Friday
 ==Friday, July 22==
 [[File:Cornell11 VioA,B,E PCR 7-22.jpg|thumb|Gel electrophoresis]]
 <br>
-Lab work done by: James Mathew & Claire Paduano
+Lab work done by: James Mathew, Claire Paduano
 <br><br>
 *PCR gel purification of VioA, VioB, and VioE
 ==Saturday, July 23==
-Lab work done by: James Mathew & Claire Paduano
+Lab work done by: James Mathew, Claire Paduano
-*Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and avitag primer dimer
+*Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and AviTag primer dimer
-__NOTOC__