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From 2011.igem.org

Contents 1 General Assembly 1.1 Day 1 1.2 Day 2 1.3 Day 3 2 Restriction Digest 2.1 Insert 2.2 Vector 3 Test Digest 4 PCR 4.1 Insert Amplification 4.2 For colony PCR 4.3 Assembly via PCR 5 Ligation 6 Transformation/Plating 7 Prepare Culture 8 Prepare Freezer Stock 9 Modified Knight Lab Multi-Site PCR 10 Modified Richard Lab Double Primer Site Directed Mutagenesis

General Assembly

Day 1

1. Plasmid extraction through miniprep
2. Insert PCR
3. Restriction digest
4. Ligation
5. Transformation
6. Plate
7. Incubate overnight

Day 2

1. Colony PCR
2. Run gel
3. If gel yields positive results, culture
4. Incubate in shaker overnight

Day 3

1. Extract modified plasmid through miniprep
2. Send plasmid to sequencing center
3. Examine sequencing results

• If sequencing results are positive, make freezer stock
• If sequencing results are negative, start again from insert PCR

Restriction Digest

Based on BioBrick Assembly Manual

Insert

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

Vector

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

Test Digest

• 4.5μl water
• 10μl miniprep
• 2μl buffer
• 2μl 10 X BSA
• 0.75μl each enzyme (either EcoRI and XbaI or SpeI and PstI)
• Incubate at 37°C for 15 minutes.

No 80°C heat kill necessary

PCR

Insert Amplification

• 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

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

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/Plating

Note: This is a procedure for performing # transformations

1. Prepare #ml of SOC medium by adding MgCl2 and Glucose to SOB medium. Add 5μl MgCl2 solution and 10μl 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 in ice. No ice water should EVER enter the cuvette.
5. Remove # tubes of competent cells from the -80°C 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 37°C bath for 1-2 hours.
12. Plate 100μl of this recovery solution on the correct selective agar plate.

Prepare Culture

• 5 ml SOB medium
• 5 μl antiobiotic
• colony stab (pipette up and down)

Prepare Freezer Stock

1. Add 300 μl overnight culture to 2 separate microcentrifuge tubes. LABEL THEM!
2. Add 300 μl of 40% glycerol to each tube.
3. Store one copy of each part in both -20° and -80° freezers.

Modified Knight Lab Multi-Site PCR

PCR Mixture

• 2.5 μl 10x Taq ligase buffer
• 2.5 μl T4 ligase buffer
• 5 μl phusion polyermerase buffer
• 1 μl dNTPs
• 1 μl K316007 miniprep
• 0.3 μl primer 1
• 0.3 μl primer 2
• 0.3 μl primer 3
• 10.2 μl dH₂O
• 0.5 μl phusion polymerase
• 1 μl T4 PNK
• 1 μl Taq ligase

The primers used for XylE mutagenesis were designed Wednesday, May 25.

PCR cycles

Temperature (°C)	Time at each temperature
37	30 minutes
95	3 minutes
95	1 minute
55	1 minute
65	extension time (*dependent on length of part)
4	hold

*Polymerase traverses 1 kbp every 30 seconds.

Modified Richard Lab Double Primer Site Directed Mutagenesis

PCR Mixture

• 36μl dH₂O
• 0.5μl template plasmid
• 10μl Phusion polymerase primer
• 1μl forward primer
• 1μl reverse primer
• 1μl DNTP
• 0.5μl Phusion polymerase

PCR Reaction

Temperature (°C)	Time at each temperature
98	30 sec
98	10 sec
60	30 sec
72	extension time (*dependent on length of part)
72	5 min
4	hold

*Polymerase traverses 1 kbp every 30 seconds.

After PCR

1. Purify PCR product
2. DpnI Digest (2 hours)
3. heat kill DpnI at 80°C
4. purify through Omega Bio-Tek cycle pure kit
5. transform into electrocompetent Escherichia coli cells
6. plate