Wayne:Laboratory Protocols

Lab safety

General guidelines:

• Always use gloves when working in the lab but DO NOT WEAR GLOVES OUTSIDE OF THE LAB (e.g. hallway, elevator, etc).
• Pre-PCR is carried out in room 4146. All post-PCR activity takes place in room 4162. Discard gloves when moving between rooms and keep all equipment in their designated rooms. AVOID moving things back into the post-PCR room 4162. These precautions are to avoid contamination problems.
• When stock solutions, kits, gloves, Kimwipes, etc runs out, it is your responsibility to make sure these get replaced. If more needs to be ordered, please notify Sarah.
• At the end of the day, remove all of your stuff from the bench you were using and return all equipment to their appropriate places, washing glassware with deionized water and hanging on the rack to dry.

Handling hazardous materials

Be extra careful when handling reactions or waste containing hazardous chemicals:

• Polyacrylamide is a neurotoxin and wear gloves. As a liquid it is particularly nasty. Wear nitrile gloves when using it, as acrylamide goes through regular latex and vinyl gloves.
• Ethidium Bromide (EtBr) is carcinogenic and wear gloves. Any waste or contaminated material (including stained gels) must be placed in the labelled containers under the UV camera in room 5318.
• Always work in the hood when handling organic solvents, like phenol and chloroform, and place waste in appropriately labelled containers.
• Formamide is carcinogenic and wear gloves. Be careful not to breathe near it, as it aspirates easily.

Sending microsatellite products to the Core facility

1. Thaw 1ml of ABI HiDi (Formamide).
2. Prepare mix of HiDi and size standard (Liz 500).

• 9.7μL HiDi and 0.3μL Liz PER SAMPLE.
  
• 1ml HiDi and 15μL Liz PER 96 SAMPLES.
  

3. 9.5μL of HiDi/Liz per well in a 96-well plate.
4. Make a 1/20 dilution of PCR product.

• 2μL PCR product to 38ul water.
  
• Use a dilution plate.
  

5. Add 2μL of diluted PCR to the 9.5ul HiDi/Liz in each well.
6. Use a sticky lid and centrifuge.
7. Denature at 95°C for 5min (disable heated lid); DENATURE program.

• Place on ice immediately for 5min.
  

8. Spin down plate and place back on ice.
9. Label plate with name: Wayne_r_<PlateName>, your initials, date.
10. Carry on ice to the Core facility (5th floor Gonda, 5309).
11. Place plate in the bottom shelf of the genotyping refrigerator labeled “Ready to Run with ABI 3700”.
12. Plate results will be on the WebSeq webpage, to be genotyped using GeneMapper.

Ethanol precipitation for DNA clean-up

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.

Phenol/Chloroform extraction of genomic DNA

Day 1: Digestion of Sample
1. Finely mince ~25 mg of tissue or 200 µl of blood to a 2.0 ml tube.
2. Add the following reagents to the tube:

• 500 µl of 1x TEN Buffer
  
• 50 µl of 10% SDS
  
• 40 µl of Proteinase K (20 mg/ml)
  

3. Mix by vortexing
4. Incubate at 56 °C overnight on a rocking platform.

Day 2: PCI Extraction

• Make sure the relevant buffers (PCI, CI) and reagents (NaOAc, 70% EtOH) are prepared!
  

1. Remove tubes from incubator, and add 500 µl of buffered phenol. Shake tubes and invert several times (5 – 10 mins). Spin tubes in centrifuge at max speed (14,000 rpms) for 5 mins.
2. Observe 2 layers. The top layer contains the DNA/RNA and the bottom, organic layer the waste.
3. Remove the top layer and transfer into a new and labeled tube, and discard the bottom layer in the PCI organic waste container.
4. Add 500 µl of PCI to the samples in the new tubes, and shake tubes and invert several times (5 – 10 mins). Spin tubes in centrifuge at max speed (14,000 rpms) for 5 mins.
5. Observe 2 layers. The top layer contains the DNA/RNA and the bottom, organic layer the waste.
6. Remove the top layer and transfer into a new and labeled tube, and discard the bottom layer in the PCI organic waste container.
7. Add 500 µl of CI to these new tubes, and shake tubes and invert several times (5 – 10 mins). Spin tubes in centrifuge at max speed (14,000 rpms) for 5 mins.
8. Pipette off the top layer and transfer it to a 1.7ml final epi tube with the final sample name and number.
9. [OPTIONAL] If you want to RNase treat the DNA, add 1ul of a 10 µg/mL stock solution of RNase A to your DNA and incubate at 37ºC for 30-60 min.
10. To this tube add the following:

• 100 µl of 3M sodium acetate (NaOAc)
  
• 1 ml of cold 100% ethanol (EtOH)
  

11. Invert tubes several times and place in a -20 freezer overnight. (3 hours is sufficient).
12. You can either stop here and do precipitation the next day or after 3 hours, complete the precipitation.

Ethanol Precipitation
1. Remove tubes from the freezer, and centrifuge at max (14,000 rpms for ~10 min) to pellet the DNA.
2. Observe a white or brownish pellet. (May be absent in low concentration samples).
3. Decant the supernatant, being careful not to lose the pellet.
4. Add 1ml of fresh 70% EtOH to the tube containing the pellet. Vortex briefly to re-suspend the pellet.
5. Centrifuge at max (14,000 rpms for 10 min) to pellet the DNA.
6. Decant the EtOH, being careful not to lose the pellet.
7. Remove the remaining 70% EtOH by vacuum centrifuging in the tubes in the Savant Speed-Vac for 10 mins or until the pellet is dry. (Do not use high heat for more than 5 mins).
8. Re-suspend DNA in 100-200 1x TE (or Qiagen’s AE) Buffer.

RNase treatment [if you didn’t do this prior to precipitation]

• If you are extracting DNA from an RNAbuffer (e.g. PAXgene RNA tubes which do not contain any DNases), you may consider doing an RNAse treatment of the final DNA*
  

1. Add 1ul of a 10 µg/mL stock solution of RNase A to your DNA and incubate at 37ºC for 30 min.
2. Recover the DNA by adding 1/10 volume of 3M sodium acetate (pH 6.8) and 2 volumes of isopropanol or 95% ethanol to the DNA containing solution.
3. Incubate on ice for 10 min.
4. Centrifuge at maximum speed for 5 min at room temperature, to pellet the DNA.
5. Discard (carefully) the alcohol. Wash with 70% ethanol and dry DNA via Speed-Vac on low heat for 10 mins.
6. Dissolve in AE, TE or dH₂O, as you did in the DNA extraction. 



Common buffers and preparations by the final desired volume:

TEN Buffer 1x	50mL	25mL
0.5M EDTA	100μL	50μL
5M NaCl	1mL	500μL
1M TRIS	500μL	250μL
ddH2O	48.41mL	24.2mL

PCI	50mL	25mL	12.5mL
Buffered Phenol	25mL	12.5mL	6.25mL
Chloroform	24mL	12mL	6mL
Iso-Amyl Alcohol	1mL	500μL	250μL

CI	50mL	25mL
Chloroform	48mL	14mL
Iso-Amyl Alcohol	2mL	1mL

70% Ethanol	50mL
100% Ethanol	35mL
ddH2O	15mL