"
BioBrick Protocol
PCR and tube contents -Extract insert from the genome through the use of overhang PCR.
1. In a microcentrifuge tube mix the following reagents for a Herculase PCR.
| Reagents | 1X Volume uL | RX Volume uL |
| Herculase 5X Buffer | 10 | |
| 2.5mM dNTP Mix | 5 | |
| 100-300ng genomic DNA | X | |
| 10uM Forward Primer | 1.25 | |
| 10uM Reverse Primer | 1.25 | |
| Herculase II Enzyme | 1 | |
| Sterile Water | Y | |
| Total | 50 |
| Temperature | Time | Cycles |
| 95 Celsius | 2 minutes | 1 |
| 95 Celsius | 20 seconds | 30 |
| 55 Celsius | 20 seconds | 30 |
| 72 Celsius | 30 seconds | 30 |
| 72 Celsius | 3 minutes | 1 |
2. In a separate tube mix 1uL 6Xloading dye and 5uL DNA.
3. Run sample on a gel with 6uL 2 log ladder.
4. PCR purify the inserts and elute with 10mM Tris HCl at ph7.4.
5. Digest the inserts and a sample of RFP vector with EcorI and SpeI. Table below shows a 1X reaction.
| Reagent | 1X Volume uL | RX Volume uL |
| NEB Buffer 4 | 3 | |
| Insert | 25 | |
| EcorI | 1 | |
| SpeI | 1 | |
| BSA | .3 |
6. Allow digestion to run for at least 1 hour at 37 Celsius heater.
7. Heat inactivate enzyme at 60 Celsius heater for 20 minutes.
8. Load all 30uL on a gel (100mL TTE, 1g Agarose; after cooling add 10uL EtBr).
9. Run gel at 140volts for about 30 minutes.
10. Take a picture of gel quickly to see how the bands ran.
11. Gel extract pieces. (Note: RFP will split into two bands, the one at 2000kb is the backbone)
12. Gel purify the pieces through the use of Qiagen Gel Extraction Kit.
13. Elute inserts with 20uL Tris HCl at pH7.4.
14. Elute backbone with 40uL Tris HCl at ph7.4.
15. Prepare ligation mixture.
^ Reagent ^ 1X Volume uL ^ RX Volume uL ^ ^ 10X T4 Lig. Buffer | 1.5 | | ^ Insert | 6(hold off) | | ^ Vector | 3 | | ^ T4 Ligase | 1 | | ^ sterile water | 3.5 | |
16. Aliquot into as many tubes as necessary.
17. Add the inserts to the aliquoted tubes.
18. Set ligation to run overnight at 16 Celsius or at room temperature for an hour.
19. Thaw competent cells for 15 minutes on ice.
20. Pipette into individual tubes per inserts ligated.
21. Add 5uL of ligation product.
22. Flick 10-20 times to ensure plasmid is mixed with cells.
23. Heat shock cells for 45 seconds and then put on ice for 2 minutes.
24. Resuspend cells in 450uL LB and place in shaker for an hour.
25. Spread cells on chloroamphenicol plates.
26. Allow plates to grow overnight at 37 Celsius.
27. Pick 8- 10 Colonies 12-16 hours later and grow up in LB with chloroamphenicol at 34ug/mL.
28. Pipette 2uL sample of colonies into 50uL sterile water.
29. Heat in PCR machine to 95 Celsius for 10 minutes.
30. Place lysed sample on ice for 5 minutes.
31. Prepare csPCR master mix.
^ Reagents ^ 1X Volume uL ^ Rx Volume uL^ ^ Buffer | 10 | | ^ 2.5mM dNTP Mix | 5 | | ^ Plasmid | 4 (hold off) | | ^ 10uM F. Sequencing Primer | 1.25 | | ^ 10uM R. Sequencing Primer | 1.25 | | ^ rTaq Polymerase | 1.25 | | ^ Sterile Water | Y | | ^ Total | 25 | |
32. Aliquot into individual tubes.
33. Add boiled DNA to each tube.
34. Run PCR at the following temperatures and times.
^ Temperature ^ Time ^ Cycles ^ | 95 Celsius | 2 minutes | 1 | | 95 Celsius | 20 seconds | 30 | | 55 Celsius | 20 seconds | 30 | | 72 Celsius | 30 seconds | 30 | | 72 Celsius | 7 minutes | 1 |
35. Run gel (100mL TTE, 1% agarose, 10uL EtBr when cooled).
36. Take a picture and analyze.
37. Send correct colonies for sequencing.
38. Two tubes are required for each plasmid to be sequenced, add 500ng of DNA per tube.
39. Fill up to total of 10uL with sterile water.
40. Add 5uL of 5mM Forward to one tube.
41. Add 5uL of 5mM Reverse to other tube.
42. Repeat with each DNA piece to be sequenced.
