Wayne:Laboratory Protocols: Difference between revisions
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Ethanol precipitation of DNA: Primarily used to clean-up DNA that has been stored in phenol<br> | Ethanol precipitation of DNA: Primarily used to clean-up DNA that has been stored in phenol<br> | ||
1. In the 1.5mL epi tube containing the DNA sample to clean, add 1mL of COLD 100% ethanol and 100ul sodium acetate (NaOAc) pH 5.2. Mix by inverting<br> | 1. In the 1.5mL epi tube containing the DNA sample to clean, add 1mL of COLD 100% ethanol and 100ul sodium acetate (NaOAc) pH 5.2. Mix by inverting.<br> | ||
2. Place in -20°C freezer for at least 3 hours, overnight is fine<br> | 2. Place in -20°C freezer for at least 3 hours, overnight is fine.<br> | ||
3. Spin at 14,000 rpm in a chilled centrifuge at 4°C for 10-15 min<br> | 3. Spin at 14,000 rpm in a chilled centrifuge at 4°C for 10-15 min.<br> | ||
4. Discard supernatant. Careful not to disturb pellet! Pellet may be invisible<br> | 4. Discard supernatant. Careful not to disturb pellet! Pellet may be invisible.<br> | ||
5. Re-suspend in 1mL fresh 70% ethanol, centrifuge for 5min at 14,000 rpm<br> | 5. Re-suspend in 1mL fresh 70% ethanol, centrifuge for 5min at 14,000 rpm.<br> | ||
6. Discard supernatant<br> | 6. Discard supernatant.<br> | ||
7. Dry pellet in Speed Vac for up to 10min at low or medium heat<br> | 7. Dry pellet in Speed Vac for up to 10min at low or medium heat.<br> | ||
8. Re-suspend pellet in 1x TE or Qiagen AE buffer. Generally use 50-200ul, depending on amount of DNA present. Thermomixer is probably ideal at RT.<br> | 8. Re-suspend pellet in 1x TE or Qiagen AE buffer. Generally use 50-200ul, depending on amount of DNA present. Thermomixer is probably ideal at RT.<br> | ||
Revision as of 15:28, 31 January 2013
Put protocols here for:
msats
lab safety
sequencing
pcr
primers
Ethanol precipitation of DNA: Primarily used to clean-up DNA that has been stored in phenol
1. In the 1.5mL epi tube containing the DNA sample to clean, add 1mL of COLD 100% ethanol and 100ul sodium acetate (NaOAc) pH 5.2. Mix by inverting.
2. Place in -20°C freezer for at least 3 hours, overnight is fine.
3. Spin at 14,000 rpm in a chilled centrifuge at 4°C for 10-15 min.
4. Discard supernatant. Careful not to disturb pellet! Pellet may be invisible.
5. Re-suspend in 1mL fresh 70% ethanol, centrifuge for 5min at 14,000 rpm.
6. Discard supernatant.
7. Dry pellet in Speed Vac for up to 10min at low or medium heat.
8. Re-suspend pellet in 1x TE or Qiagen AE buffer. Generally use 50-200ul, depending on amount of DNA present. Thermomixer is probably ideal at RT.
