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From 2011.igem.org

Contents 1 Restriction Digest 1.1 Insert 1.2 Vector 2 PCR 2.1 Create Insert 2.2 For colony PCR

Restriction Digest

Insert

• 38ul water
• 5ul buffer
• 5ul Purified PCR product
• 1ul BSA
• 1ul each enzyme (either EcoRI and SpeI or XbaI and PstI)
• 0.5ul DpnI
• Put in 37C water bath for 1-2 hours
• Heat kill for 20 mins in 80C bath

Vector

• 32ul water
• 10ul miniprep
• 5ul buffer
• 1ul BSA
• 1ul each enzyme (either EcoRI and XbaI or SpeI and PstI)
• Put in 37C water bath for 1-2 hours
• Add 6ul Antarctic Phosphatase Buffer and 1ul AP
• Incubate for another hour at 37C
• Heat kill for 20 mins in 80C bath

PCR

Create Insert

• 42ul water
• 5ul buffer
• 1ul DNTPs
• 1ul Forward Primer
• 1ul Reverse Primer
• 0.5ul VENT polymerase
• 0.5ul template DNA (plasmid from miniprep or registry)

     Run thirty cycles of the "Taq Vent PCR" program but adjust the extention time to fit your part. Extention time is based on the size of ...your part. Vent polymerase proceeds at 1kb/min. Once you know your part size, add 300bp and this will determine your extention time. The minimum extention time is 45 seconds. It's also good to give a little extra extention time.      Examples: part is 200 bp, extention time of 45 seconds; part length of 700bp, extention time of 1min:10secs, part length of 1.6kb, extention time of 2min.

For colony PCR

• 42ul water
• 5ul buffer
• 1ul DNTPs
• 1ul Forward Primer
• 1ul Reverse Primer
• 1ul TAQ polymerase
• Colony stab (template DNA)

     To get template DNA just stab a colony with a pipette tip and then pipette up and down in the PCR mix. Run the "Colony PCR" program, and adjust your extention time as described above.