Human Bocavirus (HBoV) RT-PCR
Two-Step PCR Set up
Date: ________________
Initials: ________________
1. Label 0.2ml thin-walled strip tubes with ID # and put on ICE.
2. Add 4ul of random decamers (50 μM) to each tube (can use the same tip for all tubes).
Lot#________________
Exp date__________________
3. Pipet 22ul of sample and/ or control into the appropriate tube, mix by pipetting.
4. Keep tubes on ice until they are placed into the thermocycler.
5. Run the samples under the following conditions:
80ºC 3 min.
4 ºC ∞
6. Keep samples on ice after removing from thermalcycler.
7. Make the Master Mix by combining the reagents listed below on ice. The reagent volume is
multiplied by n = the number of samples + 1.
Step 1 RT Reaction Mix
Added
TOTAL
1 Rxn
7.7 l
4.0 l
0.8 l
0.5 l
1.0 l
Total Rxn
7.7 * n
4.0 * n
0.8 * n
0.5 * n
1.0 * n
14.0ul
14.0 * n
Reagent
dH20 (Gibco Ultra Pure)
10X RT Buffer
dNTP (25mM)
Lot #
Exp.
Ambion RNase Inhibitor 40 U/ul
Ambion M-MLV-Reverse
Transcriptase (100 U/ul)
8. Add 14.0 l of RT Rxn Mix into each tube containing sample and decamer. Mix by
pipetting up and down. Change pipet tips between samples.
9. Quick spin tubes to remove air bubbles and put tubes into thermocycler.
10. Run under the following conditions.
Step
1
2
3
Temperature Time
44°C
1 hr
92°C
10 min
4°C
∞
Step Two PCR
Date: ______________
Initials: ________________
1. Label 0.2ml thin-walled strip tubes with ID # and put on ICE.
2. Calculate the appropriate amount of the PCR master mix by calculating n (n=the
number of reactions and controls plus one. Multiply the single Rxn volume by this
number.
PCR Master Mix
Added
Total
1 Rxn
38.6 l
5.0 l
0.2 l
0.2 l
1.0 l
45.0 l
5.0 l
x n = n Rxn
38.6 x n
5.0 x n
0.2 x n
0.2 x n
1.0 x n
45.0 X n
50.0 X n
Reagent
dH20 (Gibco Ultra Pure)
10X Pfu Buffer
Lot #
Exp.
Forward primer HBoV01.2 [50mM]
Reverse primer HBoV02.2 [50mM]
Pfu DNA Polymerase [1.25 units]
First strand cDNA
3. Assemble the PCR components on ice, and mix gently.
4. Aliquot 45ul of the PCR mix into the previously labeled 0.2ml strip tubes.
5. Using a multichannel pipet, pipet 5.0 l of the sample cDNA into the PCR mix and
pipet up and down to mix.
6. Spin briefly and position tubes into the thermocycler.
7. Verify the program conditions before starting the program.
Step
1
2
3
4
5
6
Date: ______________
Temperature
94°C
94°C
56°C
72°C
72°C
4°C
Time
1 min
20 sec
20 sec
30 sec
5 min
Return to Step#
# Cycles
2
45
∞
Initials: ________________
1% Agarose Gel
100 ml 1X TAE Buffer
1 gram molecular grade agarose
Bring to a boil. Cool to about 55oC. Add 1-2 µl EtBr. Stir well and pour into gel frame.
Allow gel to solidify.
Place solidified gel into a horizontal tank filled with 1X TAE. Load 7 µl ladder onto gel. Mix
2 µl 10X Blue Juice with 7 µl sample/ control. Load carefully onto gel. Run @ 120 V for
approximately 2 hours.
Start time_______________
Stop time_______________