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Revision as of 21:09, 11 August 2010

Protocols

Making Electro-Competent Cells

Materials:
• DH5alpha glycerol stock
• Two LB plates
• 5mL LB and 14ml round bottom polystyrene tube
• 500mL LB
• 1L chilled water
• 50mL conical vials
• 40mL of 10% Glycerol
• **Must reserve the table top centrifuge
Directions:
1. Streak out DH5alpha from glycerol stock; grow overnight
2. Re-streak your DH5alpha; grow overnight*
3. Pick colony and grow in 5mL LB overnight
4. Add 2.5mL of the DH5alpha overnight to 500mL of LB
5. Let grow 4-6 hours until the OD is >=0.5
6. Centrifuge 10min. at 8,000rpm at 4degC
7. Pour out supernatant and resuspend in 5mL ice-cold water. Then add 500mL cold water and mix well
8. Centrifuge 10 min. at 8,000rpm at 4degC
9. Pour out supernatant and resuspend in 5mL cold water. Then add 500mL cold water and mix well.
10. Centrifuge 10 min. at 8,000rpm at 4degC
11. Pour out supernatant and resuspend in 5mL cold water. Pour into 50mL Vial and add 40mL 10% glycerol
12. Centrifuge 10min. at 4,200rpm at 4degC.
13. Resuspend in 1mL 10% cold glycerol and mix.
14. Pour into micro-centrifuge tubes and store in -80degC
*This will remove all the glycerol from the cells.

Site Directed Mutagenesis

Procedure modified from [http://www.genomics.agilent.com/files/Manual/200555B_01.pdf Stratagene QuikChange II Protocol]

Materials

• PFU Ultra Polymerase (high fidelity)
• 10X Reaction Buffer
• DpnI (20U/μL)
• dNTPs
• Competent Cells

General Overview
1. Design primers
2. Mutant strand synthesis (PCR)
3. DpnI digestion of template
4. Transform and plate
Primer Design Considerations
• Both primers must contain desired mutation and anneal to same sequence on opposite strands of the plasmid.
• Keep the Primers aruond 25-45 bp long with 10-15 bp on either side of the mutated base.
• Keep the melting temperature greater than or equal to 78°C
Tm=81.5+,41(%GC)-675/N-%mismatch where N is the length of the primer
• Try to keep the primers to a minimum GC content of 40% and end the primer in GC for a clamp.
• Make sure primers are in excess to template
PCR

Ingredient	Amount
10X Buffer	5 μL
Template	20 ng
Primer 1	125 ng
Primer 2	125 ng
10 mM dNTPs	1 μL
Polymerase	1 μL
H2O	Bring to 50 μL

Program:

95°C	3 min
95°C	30 sec
annealing temp	1 min
72°C	1 min/Kb
go to step 2	12X
72°C	1 min
hold at 4°C

PCR Purification via Promega Kit
DpnI Digestion

DNA	500 ng
Buffer 4	5 μL
BSA	.5 μL
DpnI	1 μL
H2O	Bring to 50 μL

Incubate at 37°C for 1 hour, inactivate at 80°C for 20 min.
Transform via electroporation