(last updated 10/05/2004)

RNA Extraction (QIAgen Viral RNA Kit) (BL3)

When working in the BL3, remember to use extreme care. Clean the workspace with 70% Et-OH before and after use, and make sure that contaminated tubes and tips are neutralized with Westcodyne.

• Check the heat block. Heat to 80°C, if needed.
• Pull the samples from the freezer to thaw.

Buffer AVL precipitates at 4°C after addition of the Carrier RNA.

• Remove Buffer AVL from the fridge and place on the 80°C heating block. Vortex after ~2 minutes. Do not heat for more than 5 minutes, and do not heat more than 6 times.

Carrier RNA dissolved in buffer AVL is stable for up to 6 months at 4°C.

For each quadruple extraction:

• 1 5mL tube to contain the buffered plasma
• 2-4 cryo tubes for the extra plasma aliquots
• labels for the aliquots
• 10 collection tubes
• 1 1.5mL tube
• 1 spin column (in a collection tube)

Before using buffer AVL for the first time, check the buffer for precipitate, and incubate at 80°C, if needed, until precipitate dissolves. Add 1 mL of buffer AVL to the tube of lyophilized Carrier RNA. Dissolve the RNA thoroughly and transfer back to the buffer bottle. Mix thoroughly and aliquot 1.0mL into dated 1.5 mL tubes. Store at 4°C and use within 6 months.

Before using buffers AW1 and AW2 for the first time, add the appropriate amount of Et-OH. Mix thoroughly.

1. All buffers should be at room temperature prior to use.
2. Label the 5mL tubes.
3. Pipet 2240μL of Buffer AVL into each 5mL tube.
4. Add 560μL of the plasma sample. Mix thoroughly by pipetting. (some samples foam at this point)

If cryoprecipitates are present in the thawed plasma sample, they can be pelleted by briefly centrifuging at 6800 x g for 3 minutes. This step will not reduce viral titers.

1. Incubate at room temperature for 10 minutes.
2. Aliquot the remaining plasma, label tubes, and place in freezer.
3. After 10 minutes, add 2240μL 96% Et-OH to the 5mL tube. Mix thoroughly.
4. Remove 630μL from the 5mL tube and apply it to the spin column. Take care to not wet the rim.
5. Spin at 6000 x g for 1 min.
6. Place spin column in a clean collection tube and discard the tube containing the filtrate.
7. Repeat the previous three steps until all the buffered plasma has been applied to the column.
8. Label the 1.5mL tubes.
9. Wash the column with 500μL of Buffer AW1.
10. Spin at 6000 x g for 1 min.
11. Place spin column in a clean collection tube and discard the tube containing the filtrate.
12. Wash the column with 500μL of Buffer AW2.
13. Centrifuge at full speed for 3 minutes. (20,000 x g)
14. Place spin column in a clean collection tube and discard the tube containing the filtrate.
15. Centrifuge at full speed for 1 min.
16. Place the spin column in the labeled 1.5mL tube. Apply 40μL of Buffer AVE directly to the center of the membrane and incubate at room temperature for 1 min.
17. Centrifuge at 6000 x g for 1 min.
18. Apply another 40μL of Buffer AVE to the center of the membrane and incubate at room temperature for 1 min.
19. Centrifuge at 6000 x g for 1 min.

Place the 1.5mL tubes on foil and spray with 70% Et-OH. Make a packet with the foil and spray the outside with 70% Et-OH before removing from the BL3.

In the PCR room, aliquot 25μL of the RNA into labeled 0.6mL tubes using low retention tips.

Viral RNA is stable for up to 1 year when stored at -20°C or -70°C.

Proceed to cDNA reaction for one aliquot immediately following extraction. This avoids a freeze/thaw of our sample and may allow a greater chance of successful amplification.

Whole Genome Reverse Transcription (Invitrogen RT Kit)

1. Mix per sample and preheat RT-PCR machine to 65°C:

   	RNA	~25μL
   	dNTP (20mM)	1.5μL
   	oligo-dT (20pmol/μL)	2.5μL

2. Start file#33 - heat at 65°C for 5 min
3. Reduce heat to 45°C (higher temp will inactivate RT!)
4. Add 20μL of the following pre-warmed!!! mix per sample:

   	5x buffer	8μL
   	0.1M DTT	4μL
   	Superscriptase III	2μL
   	RNase inhibitor	1μL
   	dH2O	5μL

5. Incubate 1.5 hours at 45°C
6. Add additional 1.0μL Superscriptase III, and incubate another 1.5 hours at 45°C
7. Inactivate for 15 min at 70°C
8. Add 1μL RNase H and incubate for 20 min at 37°C
9. Label tube as cDNA and date. Freeze in Walker cDNA box.

Hot Start PCR

1st Round	Lower Premix		Upper Premix
20 mM dNTP	0.9 μL	DNA	1μL
50pmol/μL 1.U5C	0.3 μL	10X Buffer 1	5 μL
50 pmol/μL 1.U5Cb	0.3μL	Expand enzyme	0.75 μL
50pmol/μL 1.3.3plC	0.3 μL	H2O	23.25 μL
H2O	18.2 μL	Total	30 μL
Total	20 μL
μL / sample		μL/sample

2nd Round	Lower Premix		Upper Premix
20 mM dNTP	0.9 μL	1st round product	1μL
50pmol/μL 2.U5C	0.3 μL	10X Buffer 1	5 μL
50pmol/μL 2.3.3plC	0.3μL	Expand enzyme	0.75 μL
H2O	18.5 μL	H2O	23.25 μL
Total	20 μL	Total	30 μL
μL / sample		μL/sample

dNTPs final concentration: 360 μM

primers final concentrarion: 300nM

MgCl final concentration = 1.75 mM

Cycle Profile: WHOLGENM

1x	94°	2'
10x	94°	10"
	68°	30"
	68°	8'
20x	94°	10"
	68°	30"
	68°	8' + 20"/cycle
1x	68°	20'
1x	4°	forever

SET-UP (individual tubes and paraffin):

1. Place 1mL microfuge tube filled with paraffin in heat block (heated to highest temp), place p200 tip in the wax to preheat (otherwise wax will just solidify in the cold tip)
2. Make lower master mix for “x” number of reactions, keeping all reagents cold on ice.
3. Aliquot 20μL of mix to each PCR reaction tube (use 0.5mL dome-lid thin-wall PCR tubes from Island Scientific)
4. Place lower tubes in the heat block and add 25μL paraffin wax to them – the wax will melt into the correct place
5. As soon as the wax melts, transfer them to your rack to cool (the wax will form a layer over the lower reaction mix, there will be a dimple in the middle of the wax plug but there should be no opening through to your lower reaction mix)
6. Make your upper reaction master mix, keeping all reagents cold on ice.
7. Aliquot 29μL into each reaction tube on top of the wax
8. Add your sample template to each tube (upper mix)
9. Transfer the reactions to your bench to add the control DNA (use 10 copy to measure sensitivity for first and second rounds combined and a 1000 copy control for individual rounds – if you can see a 1000 copy control after a single round, you’re doing GREAT!)
10. Start the PCR machine (MJ) and allow the block to get to 80°C before starting to load your samples

Notes:

• These primers will only work on full-length virus that has both LTRs – they will NOT work on pNL4-3

SET-UP (8-well strip tubes):

1. Place 8 well ultra-thin strip tubes (Island Scientific) into 96-well cold block. Notice the numbering of the tubes and orient correctly.
2. Make lower master mix for “x” number of reactions, keeping all reagents cold on ice.
3. Aliquot 20μL of mix to each PCR reaction tube.
4. Add one Ampliwax pellet into each tube. Place strips in the heat block unti wax melts.
5. As soon as the wax melts, transfer them to your cold block to cool (the wax will form a layer over the lower reaction mix, there will be a dimple in the middle of the wax plug but there should be no opening through to your lower reaction mix)
6. Make your upper reaction master mix, keeping all reagents cold on ice.
7. Aliquot 29μL into each reaction tube on top of the wax.
8. Add your sample template to each tube (upper mix)
9. Transfer the reactions to your bench to add the control DNA (use 10 copy to measure sensitivity for first and second rounds combined (nested) and a 1000 copy control for individual rounds – if you can see a 1000 copy control after a single round, you’re doing GREAT!)
10. Start the PCR machine (PE) and allow the block to get to 80°C before inserting your samples.

PCR Gel Purification and Cloning (Invitrogen XL TOPO PCR Cloning Kit)

Gel Purification:

1. If the PCR product is several days old, add 0.5μL of Bioline biolase and incubate for 20 min at 72°C
2. Prepare 150mL 1% agarose gel TAE buffer, microwave heat for 4 min.
3. Add 90μL of 2mg/mL of crystal violet, pour the gel and put the combs which can hold all PCR(~40μL) products
4. Add 8μL of 6X crystal loading dye to 40μL PCR product
5. Load all PCR products and 4μg HindIII run 100V for around 2 hours (note: skip one lane between each sample when you load the PCR products. It helps you to cut the gel easily without contamination)
6. Cut the right size band, put into the pre-weighted 1.5mL tube and weigh (usually i t weighs 100mg=100μL)
7. Add 250μL (2.5 volumes) of 6.6M sodium iodide and incubate at 42-50°C until the agarose is completely melted
8. Add 525μL (1.5 volumes) of binding buffer and mix well
9. Load all of the mixture onto the column, spin at 3000xg for 30 sec
10. Pour the elution in the collection vial back onto the column and repeat step 9
11. Repeat step 10 one more time to bind all the DNA to the column (a total of 3 times)
12. After the last centrifugation, discard the flow-through in the collection tube
13. Add 400μL of 1X final wash (dilute the 4x wash with ethanol) to the column and spin at 3000xg for 30 sec
14. Repeat step 13 and discard the flow-through in the collection tube after the final centrifugation
15. Spin the column again at 10,000xg for 2 min to dry the column resin
16. Transfer the column to a new 1.5mL tube
17. Add 40μL of TE buffer and incubate at room temp for 1 min
18. Spin at 10,000xg for 2 min
19. Assay 5μL by EtBr agarose gel to estimate the DNA concentration
20. Proceed directly to the TOPO Cloning reaction

TOPO Cloning and Transformation:

1. Set up the following 5μL cloning reaction per sample: | | | | —————————— | —- | | gel purified long PCR products | 4μL | | pCR-XL-TOPO vector | 1μL |
2. Mix gently and incubate for 5 min at room temp
3. Add 1μL of the 6X TOPO cloning stop solution and place on ice
4. Add 2μL of the cloning reaction into a vial of pre-thawed One Shot cells and mix gently (Do not mix by pipetting up and down)
5. Incubate on ice for 30 min
6. Heat shock the cells for 45 sec at 42°C without shaking
7. Immediately transfer the cells to ice and incubate for 2 min
8. Add 250μL of SOC medium
9. Shake the tube horizontally at 37°C for 1 hour
10. Spread 150μL from the transformation on a pre-warmed LB plate containing 50μg/mL Kanamycin
11. Incubate the plate overnight at 30°C

Plasmid mini prep (QIAgen QIAprep Spin Column Miniprep)

Note:

1. After digestion, check the insert by agarose gel. Run gel at 25V overnight
2. Once you have identified the correct clone, make a glycerol stock for long term storage. 500 μL of culture plus 75 μL of glycerol. Mix well and snap freeze in ethanol/dry ice bath.

Plasmid midi prep (Sigma GenElute HP Plsmid Midiprep)

1. Re-plate glycerol stock on LB kanamycin (50μg/mL) plate. Incubate at 30°C overnight.
2. Streak a single colony out and inoculate a 3mL culture for 8-12 hours. Add this culture to 100mL of LB kanamycin (50μg/mL) media, shake overnight at 200rpm in 30°C incubator.
3. Midi prep (see kit protocol)
4. Elute in 1.5mL of dH₂O
5. To check the insert, digest 2μL of DNA by EcoRI
6. After digestion, check the insertion size by 1% agarose gel. Run gel at 25V overnight
7. Determine the concentration by OD260 (1:50 dilution)
8. Dilute to 200 ng/μL or precipitate and resuspend to 200 ng/μL.

Sequencing:

Send 10μL of 200 ng/μL plasmid DNA combined with 7.5μL of 1μM primer in a 96 well plate. This is enough for 2.5 reactions.