Preparation and Storage of reagents for diagnostic rRT PCR-Part-1

Scope

·       This SOP explains the procedure of Preparation, Aliquoting and Storage of kits, reagents and enzyme mix.                                                                                          

2. Definition

• rRTPCR- Real Time Reverse Transcriptase Polymerase Chain Reaction

3. Materials

• Materials required:

• Powder free gloves
• Aerosol resistant tips (10µl,20µl,200µl,1000µl)
• Discarding beaker
• Permant marker
• Tissue paper
• Micro centrifuge tube (0.2 ml, 0.5 ml) DNase, RNase free.
• A rack to hold microcentrifuge tubes

·       Equipments required;

o   Biosafety Cabinet Class II

o   Vortex Mixer

o   Minicentrifuge

o   Pipettes (10µl,20µl,200µl)

o   Deep freeze (-15°c to -25°c)

• List of reagents and Kits required         

1.     rRT PCR ITD kit

2.     rRT PCR Sabin VDPV kit

3.     1 M DTT                                                        

4.     Protector RNase inhibitor 40U/µl                                          

5.     Reverse Transcriptase 20U/µl                                  

6.     Taq DNA polymerase 5U/µl                            

7.     Buffer B

• rRT PCR ITD Kit:

Source

• rRT PCR kit is provided from WHO and CDC. The contents of the kit are as follows:

Reagents                          Quantity

1. Pan EV primers+Buffer A                                                      1 ml
2. Sabin multiplex primers+Buffer A                                        1 ml
3. Pan PV primers+Buffer A                                                      1 ml
4. Pan PV1 primers+Buffer A                                                    1 ml
5. Pan PV2 primers+Buffer A                                                    1 ml
6. Pan PV3 primers+Buffer A                                                    1 ml
7. RNase free dH2O                                                                   1 ml
8. Sabin multiplex control RNA                                                 lyophilized
9. PV1 control RNA                                                                   lyophilised
10. PV2 control RNA                                                                   lyophilised
11. PV3 control RNA                                                                   lyophilised
12. Buffer B (2 vial)                                                                     1 ml
13. 1 M DTT                                                                                 3.0µl

      Kits are stored at -20°C in deep freeze no DDF-2 in Pre PCR room.

4. Procedure

      (I) Aliquoting of primers:

• All the steps are performed in Biosafety Cabinet (Refer SOP of Biosafety cabinet No. 1 (Dengue)) in Pre PCR room.
• Take out the kit from deep freeze, thaw primers, mix on the vortex mixer for 30 seconds and spin in minicentrifuge for 1 minute to resuspend probe.
• Arrange Microcentrifuge tube of 0.5 ml in a rack and label it with the appropriate primer and date of aliquoting.
• 1 ml of primer in Buffer A is aliquoted in 5 vials of 200 µl each.
• Distribute 200µl of each primer in Buffer A into its corrosponding microcentrifuge tube with 200µl pipette.
• Keep distributed primer tubes at -20°c in deep freeze no.16 in Pre PCR room,

II) Preparation of Enzyme mix and aliquoting;

Materials required:

• Equipments required:

o   Biosafety cabinet Class II

o   Vortex Mixer

o   Minicentrifuge

o   Pipette(10µl,20µl,100µl)

o   Deep freeze (-15°c to -25°c)

o   Aerosol Resistant Tips (30 µl and100 µl)

o   0.5 ml microcentrifuge tube

·       Reagents required:

o   1 M DTT                                                         2.8µl

o   Protector RNase inhibitor 40U/µl                  27.6µl

o   Transcriptor RT20U/µl                                  18.0 µl

o   Taq DNA polymerase 5U/µl                          54.8µl

o   Buffer B                                                          1 ml

a)     Preparation of enzyme mix: