Molecular Systematics Laboratories
At the American Museum of Natural History
Wheeler Lab protocols:
Printable version of lab manuals are available in MS word format or PDF format.
DNA Extraction – Qiagen DNeasy kit.
PCR – Ready-to-go Beads.
Agarose gel Preparation
Gel Extraction (for double bands): Promega Wizard Kit
AMPure Cleanup: Robot or by hand.
Sequencing
CleanSeq Cleanup: Robot or by hand.
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.
4. Remove from incubator, vortex, add 200uL Buffer AL and vortex.
5. Place in heat block at 70 C for 10 minutes.
6. Add 200uL 100% Ethanol and transfer entire volume onto spin column.
7. Centrifuge at 8000 rpm for 1 minute; discard flow-through.
8. Add 500uLBuffer AW1 and centrifuge at 8000 rpm for 1 minute; discard flow-through.
9. Add 500uL Buffer AW2 and centrifuge at 13000 rpm for 3 minutes; discard flow through.
10. Place spin column on UV-crosslinked 1.5mL tube, add 200uL buffer AE. Let sit for 1 minute, then centrifuge at 8000 rpm for 1 minute. Repeat and then combine flow-throughs for a total volume of 400uL.
Recipe: 2uL DNA
21uL H2O
1uL each direction of primer (10M)
Seal with caps or wax.
Thermal Profiles:
Basic program |
|
94 C 5:00 |
|
94 C :30 |
|
50 C :30 |
can increase the length of annealing up to 1 minute |
72 C :45 |
can increase the length of extension up to 1:30 (for longer genes). |
35 cycles |
|
72 C 7:00 |
|
hold at 10 C |
|
Rock and Roll |
|
94 C 5:00 |
|
94 C :15 |
|
42 C :05 |
can increase the length of annealing up to :30 |
68 C :15 |
can increase the length of annealing up to :30 |
40 cycles |
|
72 C for 7:00 |
|
hold at 10 C |
|
Down/Up |
|||
94 C 5:00 |
|
|
|
94 C :30 |
94 C :30 |
94 C :30 |
94 C :30 |
54 C :45 |
50 C :45 |
52 C :45 |
54 C :45 |
72 C 1:30 |
72 C 1:30 |
72 C 1:30 |
72 C 1:30 |
10 cycles |
10 cycles |
10 cycles |
10 cycles |
72 C 7:00 |
|
|
|
hold at 10 C |
|
|
|
This can be useful for double bands, and you can use any range of annealing temperatures. |
|||
Recommendations:
12s: basic program, 50 C or 48 C, go as low as 46 C
16s: basic program, 50 C or 48 C
18s: basic program, 50 C or 48 C
28s: basic program, 50 C or 48 C
COI: Rock and roll, start at 48 C, go as low as 42 C
ExtA/B: Rock and roll with longer annealing/extension, start at 48 C, usually best at 42 C
Histone3: basic program, 50 C or 52 C
EF-1 alpha: basic program with longer annealing and extension, 54 C or 56 C or Down/Up.
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).
6. If you get very faint bands, clean as usual using AMPure, then concentrate product (see below).
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.
6. Place in the 55 C incubator for 10 minutes, or until the gel is dissolved.
7. Transfer entire volume onto minicolumn; let sit 1 minute.
8. Centrifuge at 13000 rpm for 1 minute; discard flow-through.
9. Add 700uL Membrane Wash Solution.
10. Centrifuge at 13000 rpm for 1 minute; discard flow-through.
11. Add 500uL Membrane Wash Solution.
12. Centrifuge at 13000 rpm for 5 minutes; discard flow-through.
13. Re-centrifuge for 1 minute with lid open.
14. Transfer minicolumn to a 1.5mL tube.
15. Add 50uL nuclease-free water (included in kit); let sit 1 minute.
16. Centrifuge at 13000 rpm for 1 minute.
17. Go directly to sequencing or re-amplify using a regular PCR protocol to concentrate product.
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.
10. When you are ready to set up your sequencing reactions,
11. Place plate on magnet for 10 minutes.
Faint band concentration:
After AMPure cleanup, place plate on magnet for 10 minutes and transfer product to a new tube or plate. Evaporate to dryness on DNA speedvac. Re-suspend in 10L H2O and proceed to sequencing reaction.
Set up in Eppendorf plate.
Recipe: 5uL cleaned PCR product
1uL Big Dye Extender Buffer
1uL 3.2 uM primer
1uL Big Dye
Seal with wax or heat seal.
Thermal profile:
Basic program |
94 C :30 |
50 C 1:00 |
60 C 4:00 |
35 cycles |
hold at 4 C |
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.
9. Place plate back on magnet for 5 minutes.
10. Transfer 33uL finished product to Optical or Eppendorf plate; briefly spin down to get rid of any bubbles and heat seal.
11. Run on ABI 3730.