Difference between revisions of "Help:3A Assembly Kit"
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===='''1. [[Help:3A_Assembly_Kit/Growing|Growing]]'''==== | ===='''1. [[Help:3A_Assembly_Kit/Growing|Growing]]'''==== | ||
<html><img src="https://static.igem.org/mediawiki/parts/d/db/3A-Assembly-Kit-Growing.png"></html> | <html><img src="https://static.igem.org/mediawiki/parts/d/db/3A-Assembly-Kit-Growing.png"></html> | ||
| − | Grow up the E. coli that contains your parts | + | Grow up the E. coli that contains your parts! Your kit includes agar stabs for Part A (BBa_J04500) and Part B (BBa_J04650). The agar stabs contain live bacteria (E. coli) that house your parts. The parts themselves belong to a plasmid which replicates within the E. coli cell. |
| + | So before working with your parts, you'll need to make sure you have enough copies of them. You can do this by growing up your E. coli from the stabs. More E.coli means more plasmids which means more copies of a part. | ||
| + | Use these agar stabs to streak out your bacteria onto an agar plates. Then from the agar plate you can pick a single colony to grow up a cell culture. | ||
===='''2. [[Help:3A_Assembly_Kit/Miniprep|Miniprepping]]'''==== | ===='''2. [[Help:3A_Assembly_Kit/Miniprep|Miniprepping]]'''==== | ||
<html><img src="https://static.igem.org/mediawiki/parts/5/59/3A-Assembly-Kit-MPTubes.png"></html>Miniprepping cell cultures to extract the DNA for your parts | <html><img src="https://static.igem.org/mediawiki/parts/5/59/3A-Assembly-Kit-MPTubes.png"></html>Miniprepping cell cultures to extract the DNA for your parts | ||
Revision as of 13:55, 30 April 2012
You've just received the 3A Assembly Kit! We hope your team will enjoy using the kit and in the process learn about synthetic biology based on standard parts and 3A assembly. Before you start please make sure to...
- check the contents of the box with the inventory, to make sure you have everything
- inspect the materials to make sure nothing has been damaged during shipping
- refrigerate the agar plates and any materials labelled as requiring storage at 4C.
- order the NEB reagent kit. You'll need to use the NEB enzymes and reagents for 3A assembly. See the [http://2012hs.igem.org/Sponsors High School Division sponsor page] for more information.
Contents
Introduction
The 3A Assembly Kit and protocol will take you through the process of 3A Assembly, and by the end you will have assembled your own composite part in the lab. The kit includes two parts: Part A (BBa_J04500) and Part B (BBa_J04650), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn red!
The protocol will take you through the following steps:
1. Growing
Grow up the E. coli that contains your parts! Your kit includes agar stabs for Part A (BBa_J04500) and Part B (BBa_J04650). The agar stabs contain live bacteria (E. coli) that house your parts. The parts themselves belong to a plasmid which replicates within the E. coli cell.
So before working with your parts, you'll need to make sure you have enough copies of them. You can do this by growing up your E. coli from the stabs. More E.coli means more plasmids which means more copies of a part.
Use these agar stabs to streak out your bacteria onto an agar plates. Then from the agar plate you can pick a single colony to grow up a cell culture.
2. Miniprepping
Miniprepping cell cultures to extract the DNA for your parts
3. Restriction Digest
Cutting the DNA for your parts and the pSB1C3 linearized plasmid backbone in a restriction digest
4. Ligation
Ligating your two parts together into the pSB1C3 linearized backbone
5. Transformation
Transforming the ligation to get your newly assembled composite part. At the end you should see red colonies!
6. Post your results!
Note: The 3A Assembly Kit also includes prepared plasmid DNA for your Parts A and B, so you can skip directly to step 3 if you'd like.