Team:Cornell/Week 9
From 2011.igem.org
(Difference between revisions)
(→Thursday, August 4) |
(→Thursday, August 4) |
||
Line 19: | Line 19: | ||
:'''Objective''' | :'''Objective''' | ||
- | ::Ligation of GFP, RFP, and | + | ::Ligation of GFP, RFP, and vioE genes with Avi-Tagged pZE12 backbone |
:'''Ligation''' | :'''Ligation''' | ||
::*''GFP + Avi-Tagged Backbone'' | ::*''GFP + Avi-Tagged Backbone'' | ||
Line 34: | Line 34: | ||
::::<u>1.0μL T4 DNA Ligase</u> | ::::<u>1.0μL T4 DNA Ligase</u> | ||
::::20μL Total | ::::20μL Total | ||
- | ::*'' | + | ::*''vioE + Avi-Tagged Backbone'' |
::::10.6μL Avi-Tagged pZE12 vector backbone | ::::10.6μL Avi-Tagged pZE12 vector backbone | ||
- | ::::4.1μL | + | ::::4.1μL vioE |
::::2.3μL H2O | ::::2.3μL H2O | ||
::::2.0μL T4 DNA Ligase Buffer | ::::2.0μL T4 DNA Ligase Buffer | ||
Line 49: | Line 49: | ||
::Ligation reaction volumes were calculated using a formulated spreadsheet (see Protocols 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 Protocols page) that is based on 100ng of vector backbone and a 3:1 molar ratio of insert:vector. | ||
:'''Reference for Inserts and Vector Backbone''' | :'''Reference for Inserts and Vector Backbone''' | ||
+ | ::RFP gene - 678bp | ||
+ | ::GFP gene - 717bp | ||
+ | ::vioE gene - 576bp | ||
+ | ::pZE12 vector - 2340bp | ||
==Friday== | ==Friday== |
Revision as of 21:38, 4 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 |
July 31st - August 6th
Sunday
Monday
Tuesday
Wednesday
Thursday, August 4
Afternoon lab work done by: Claire Paduano, Charlie Chung
- Objective
- Ligation of GFP, RFP, and vioE genes with Avi-Tagged pZE12 backbone
- Ligation
- GFP + Avi-Tagged Backbone
- 10.6μL Avi-Tagged pZE12 vector backbone
- 3.6μL GFP
- 2.8μL H2O
- 2.0μL T4 DNA Ligase Buffer
- 1.0μL T4 DNA Ligase
- 20μL Total
- RFP + Avi-Tagged Backbone
- 10.3μL Avi-Tagged pZE12 vector backbone
- 6.7μL RFP
- 2.0μL T4 DNA Ligase Buffer
- 1.0μL T4 DNA Ligase
- 20μL Total
- vioE + Avi-Tagged Backbone
- 10.6μL Avi-Tagged 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 waterbath overnight.
- Ligation reaction volumes were calculated using a formulated spreadsheet (see Protocols 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