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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 2.3 Assembly via PCR 3 Ligation 4 Transformation

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

• 42μl water
• 5μl buffer
• 1μl DNTPs
• 1μl Forward Primer
• 1μl Reverse Primer
• 0.5μl VENT polymerase
• 0.5μl 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

• 42μl water
• 5μl buffer
• 1μl DNTPs
• 1μl Forward Primer
• 1μl Reverse Primer
• 1μl 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.

Assembly via PCR

• 1μl Forward Primer
• 1μl Reverse Primer
• 1μl DNTPs
• 1μl TAQ polymerase
• 1μl Thermopol buffer
• 41μl dH₂O

Ligation

• 12ul water
• 2ul T4 Buffer
• 3ul Vector Digest
• 3ul Insert Digest
• 1ul T4 Ligase (add this last)
• Incubate for 10 min at room temp
• Heat kill for 10 mins at 70-80C

Note: Before you start be sure to quick centrifuge the digests to get any condensation to the bottom of the tube; otherwise you are concentrating things that shouldn't be concentrated (i.e. salts) and this will have a negative effect on transformation efficiency.

Transformation

Note: This is a procedure for performing # transformations

1. Prepare #ml of SOC medium by adding MgCl2 and Glucose to SOB medium. Add 5ul MgCl2 solution and 10ul Glucose solution per ml of SOB.
2. Distribute 900ul of SOC into # clean tubes (these are the recovery tubes, label them).
3. Fill ice bucket with ice and water
4. Add # cuvettes to the float and put it i...n ice. No ice water should EVER enter the cuvette.
5. Remove # tubes of competent cells from the -80C freezer and place in the ice float. Do everything you can to keep these cells cold.
6. Set electroporator to 1500 volts.
7. Important: Quickly centrifuge the ligations to remove any condensation.
8. Add 5ul of ligation to each tube of competent cells.
9. Quickly transfer the cell/ligation mix to the cuvette, dry, and electroporate.
10. Quickly remove the cells with a pipette, and place them in the correct recovery tube.
11. Once all cells are transformed, place the recovery tubes in the 37C bath for 1-2 hours.
12. Plate 100ul of this recovery solution on the correct selective agar plate.