MIT Synbio:IAP

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From the Cambridge 2010 iGEM team

Overview

2 Intro classes: 1 day with two info sessions for people to come and see if they want to take the class or not

• Take place before EHS training

Class 0: for people who haven't taken 7.012: DNA What is synthetic biology

• things that have been done in the past
• potential projects
• Circuits
• what you can do.

What are biobricks

• registry

circuit concepts, circuit design (and/ or gates, NOR gates, etc.)/ feedback iGEM
What they're doing: bioluminescence

Techniques to teach:

• Lab tour: pipetting, sterile
• (Minipreps)
• We need to make plates, gels
• Labeling

1. Digestion: 1.5 hr 2. Ligation: 1.5 hr, do at the end 3. Gel extract + nanodrop: 20 min prep, 30 min run; 10 min cut, 20 min spin, 5 min nanodrop: 1.5 hr 4. Transformation: 1 hr, 1 hr grow, 20 min plating 5. Picking colonies 20 min--> cell stock--> grow up (us) 6. Miniprep: 1.5 hr, nanodrop 5 min 7. Restriction map

Protocol

Restriction Digest

Recall from your lecture on Biobrick assembly that the cut sites EX-SP surrounding the desired gene allow for restriction enzymes to create sticky ends that can then be used to ligate parts together. In this lab, we will be using the restriction enzymes XbaI, SpeI, and PstI to cut our plasmid DNA.

Promoter Digestion			Protein Digestion
Component	Volume (uL)		Component	Volume (uL)
Plasmid	15		Plasmid	15
Buffer			Buffer
Water			Water
BSA			BSA
SpeI			XbaI
PstI			PstI
Total			Total

• Receive 2 tubes of DNA from group leaders. One contains your promoter plasmid, and the other your protein plasmid. They each contain 15uL of DNA in solution.
• You will do all calculations before you begin digestions. First, find the concentration of your DNA. Each tube is different. Then, knowing the concentration of your DNA is in ug/mL, calculate the ug of DNA in your tube.
• You will use 10 units of enzyme per ug of DNA in your tube. Use the table below to calculate how many uL of enzyme you will use to digest your DNA.

Enzyme	Concentration (units/mL)
EcoRI-HF	20,000
XbaI	20,000
SpeI	10,000
PstI	20,000

h1. Antarctic Phosphatase Treatment of Backbone

h3. Treatment Protocol

Antarctic Phosphatase treatment is done to phosphatase the backbone used for plasmid ligations. This is done to reduce the self ligation of a backbone digested with enzyme(s) creating compatible sticky ends and hence enhance the S/N ratio of transformations.

1. Use 5 units of Antarctic phosphatase per 1µg of DNA (over digesting by factor of X)
2. Calculate volumes
   • DNA µg = amount of DNA in backbone digest = X
   • Enzyme volume = X/5 uL (minimum of 1 uL)
   • Buffer is dilution factor x dilution of the total volume.
3. Order of filling
   • Backbone Digestion
   • Antarctic Phophatase Buffer
   • Antarctic Phosphatase
4. Incubate for 15 minutes at the specified temperature for the enzyme (37C).
5. Heat Inactivate for 5 minutes at 65C.
6. Keep the buffer on ice and the antarctic phosphatase in the benchtop coolers when on the bench.

h3. CIP Treatment Setup

Enzyme: Antarctic Phosphatase Reference: [1] Concentration: 5 U/uL Volume of enzyme required = Amount of DNA digested / 5 uL (Minimum 1 uL)

|| Component || Volume || | Backbone Digestion | Y uL | | Antarctic Phosphatase | Z uL | | NEB Antarctic Phosphatase Buffer | (Y+Z)/9 uL |

h3. Cycle Setup

1. 37C for 15 minutes.
2. 65C for 5 minutes.
3. Hold at 10C.