Team:GeorgiaTech/Protocols

From 2010.igem.org

PROTOCOLS

Preparing 1% Agarose Gels:

1. Added 180 mL 1x TBE and 1.8g agarose
2. Heated up until agarose was no longer visible
3. Let cool (approx. 10 min)
4. Added 180 սL Ethidium bromide (1000X)

PCR Purification

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)
2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).
3. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.
4. Discarded flow-through. Placed the column back in the same tube.
5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.
6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.
7. Placed the column in a clean 1.5 mL microcentrifuge tube.
8. To elute DNA, 50  µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Heat shock transformation of the plasmids into our bacteria

1. Left cells and ligation reaction products on ice.
2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).
3. Left cells on ice for 30 min.
4. Applied heat shock of 45 seconds in 42C bath.
5. Put tubes on ice for 2 min.
6. Added 250 սL of LB (room temp.)
7. Incubated 1 hour at 37 C
8. Plated 100սL and left plate in the 37 degrees incubator  
9. Incubated overnight at 37C.

Gel Extraction

1. Excise DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).
3. Incubate at 50ºC for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2 – 3 min during the incubation.
4. After the gel slice has completely dissolved, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 mL collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
8. Discard flow-through and place QIAquick column back in the same collection tube.
9. Recommended: Add 0.5 mL of Buffer GQ to QIAquick column and centrifuge for 1 min.
10. To wash, add 0.75 mL of Buffer PE to QIAquick column and centrifuge for 1 min.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
12. Place QIAQuick column into a clean 1.5 mL microcentrifuge tube.
13. To elute DNA, add 50  μL of Buffer EB (1 mM Tris-Cl, pH 8.5) or water (pH 7.0 – 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30  μL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
14. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Plasmid DNA Purification with Mini Prep Kit

1. Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4 - 6 times.
3. Add 350 µL Buffer N3 and mix immediately and  thoroughly by inverting the tube 4 – 6 times.
4. Centrifuge for 10 mins at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge.
5. Apply the supernatants from step 4 to the spin column by decanting or pipetting.
6. Centrifuge for 30 – 60 secs. Discard the flow-through.
7. Recommended: Wash the spin column by adding 0.5 mL Buffer PB and centrifuging for 30 – 60 secs. Discard the flow-through.
8. Wash spin column by adding 0.75 mL Buffer PE and centrifuging for 30 – 60 secs.
9. Discard the flow-through, and centrifuge for an additional 2 min to remove residual wash buffer.
10. Place the column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 30 µL autoclaved water to the center of each spin column, let stand for 1 min, and  centrifuge for 1 min.