Section 9 Protocols
CleanSeq Cleanup: Robot or by hand.
1. Add 10uL CleanSeq directly to sequencing reaction product.
2. Add 37.5uL 85% Ethanol and mix.
3. Place plate on magnet for 5 minutes.
4. Flip plate while still on magnet and discard ethanol.
5. Add 100uL 85% ethanol and let sit for 30 seconds
6. Flip plate while still on magnet and discard ethanol.
7. Take plate off magnet and let air dry for 10 minutes.
8. Add 40uL 0.5mM EDTA and let sit for 5 minutes.
Set up in Eppendorf plate.
5uL cleaned PCR product
1uL Big Dye Extender Buffer
1uL 3.2 uM primer
1uL Big Dye
Seal with wax or heat seal.
94 C :30
50 C 1:00
60 C 4:00
hold at 4 C
AMPure Cleanup: Robot or by hand.
1. Transfer 18uL AMPure to an Eppendorf plate.
2. Add 10uL PCR product and mix.
3. Let sit 5 minutes.
4. Place on magnet, let sit 5-10 minutes.
5. Remove entire volume, leaving beads, discard liquid.
6. Add 200uL 70% Ethanol, let sit 30 seconds, flip while still on magnet to remove ethanol.
7. Repeat Ethanol wash.
8. Take plate off magnet; let air dry for 12-25 minutes.
9. Add 40uL H2O, mixing well, to re-suspend the DNA.
Gel Extraction (for double bands): Promega Wizard Kit
1. Using the small gel rig, pour a 1% low melting point agarose gel (0.5 g in 50mL 1XTBE, 1uL ethidium bromide).
2. Add 5uL of loading dye to your PCR and load the entire volume onto the gel.
3. Run at 70 volts for 1-1.5 hours.
4. Protecting yourself from the UV, and using a razor blade or spatula, excise the band you want and put it in a 1.5mL tube.
5. Weigh each tube and add 10uL of Membrane Binding Solution per 10mg or gel.
Agarose gel Preparation
1. To make a gel, add 8-10g agarose to 400mL 1XTBE buffer.
2. Microwave for 5 minutes, pour into gel tray, let cool 30 min.
3. Add SYBRsafe stain (0.1uL per lane) to loading dye (3L per lane). Remember to add SYBRsafe stain to ladder.
4. Use 2uL of your PCR product and 3uL loading dye to check amplifications on the gel.
5. If you get double bands consistently, do a gel extraction (see below).
PCR – Ready-to-go Beads.
Recipe: 2uL DNA
1uL each direction of primer (10M)
Seal with caps or wax.
94 C 5:00
DNA Extraction – Qiagen DNeasy kit.
Note the number of spin columns you use.
The centrifuge can hold up to 30 samples at once.
Forceps are to be sterilized in Fine Science Tools heat block at >200 C for>20 seconds.
1. Isolate a suitable piece of tissue and place in a UV-crosslinked 1.5mL tube.
2. Add 180uL Buffer ATL and 20uL Proteinase K and vortex.
3. Place in the 55 C incubator for 3 hours or overnight.