Preparation and Storage of reagents for diagnostic rRT PCR-Part-2

Procedure:

o   All the steps are performed in Biosafety Cabinet (Refer SOP of Biosafety cabinet No. 1(Dengue)) in Pre PCR room.

• Take out the above reagents from deep freeze, thaw them, and mix on the vortex mixer for 30 seconds and spin in minicentrifuge for 1 minute to clear the droplets from the lid of vial.
• To make 1 ml enzyme mix, add following reagents in 1 ml Buffer B vial in the below mentioned order with RNAase and DNAase free filter tips:

§  1 M DTT                                              2.8µl

§  Protector RNase inhibitor 40U/µl      27.6µl

§  Transcriptor RT20U/µl                      18.0µl

§  Taq DNA polymerase 5U/µl              54.8µl

o   Use separate filter tips for each reagent.

o   Vortex vial properly and label as +E and date of preparation.

b)                                 Aliquoting of Enzyme mix:

• Arrange microcentrifuge tube of 0.5 ml and Label each with +E and date of preparation.
• Make 10 aliquotes of 100µl each.
• Distribute 100µl of +E in to microcentrifuge tube with 100µl pipette.
• Store aliquates in deep freeze at -20°C in deep Freeze no.DF-6 in pre PCR room

5. Safety conditions

• The ulmost care should be taken to avoid cross contamination of primers during aliquoting of primers.
• Care should be taken to avoid aerosol creation during aliquoting of primers and spilled droplets must be immediately cleaned with 1% Sod. Hypochlorite solution.

6. Documentation

·       Log book of Biosafty Cabinet No.1(Dengue) PR-47

·       rRT PCR reagent Register PR-53

7. References used to draw SOP

• Manual On Intratypic differentiation of Polioviruses by Real time RT-PCR of CDC
• WHO guidelines
• Kit Insert

. Scope

·       This SOP explains the procedure of Preparation, Aliquoting and Storage of positive control RNA.                                                                                                                                                                                                          

2. Definition

• rRTPCR- Real Time Reverse Transcriptase Polymerase Chain Reaction  

Preparation of positive control RNA:

o   Positive control RNA vials are provided in lyophilized form in rRTPCR kit (ITD and VDPV kit) and are to be hydrated with dH2O provided in the kit.

3. Materials

• Equipments required

• Biosafety Cabinet Class II
• Vortex Mixer
• Minicentrifuge
• Pipette (200µl)
•  Deep freeze (-15°c to -25°c)

• Materials required

o   Positive control RNA vials(PV1, PV2, PV3 vials) lyophilized

o   dH2O provided in the kit

o   Powder free gloves

o   Aerosol resistant tips (10µl,20µl,200µl)

o   Discarding beaker

o   Tissue paper and permanent marker

o   Micro centrifuge tube (0.2 ml, 0.5 ml) DNase, RNase free.

o   Rack to hold microcentrifuge tubes

4. Procedure

·       All the steps are performed in Biosafety Cabinet (Refer SOP of Biosafety cabinet 7) in PCR room.

·       Take out the Positive control RNA vials from the kit.

·       Thaw dH2O provided in the kit.

·       Briefly spin the tubes to concentrate the lyophilized pellets before resuspention.

• Add 100µl dH2O with sterile RNAase free tip in each lyophilized control to get working solution of 10 pg /µl.

• After addition of dH2O allow the vial to settle at room temperature for 10-15 min.

      for proper rehydration.

• Arrange 4 microcentrifuge tubes of 0.2ml for each RNA control. Label as PV1, PV2, PV3 and date of preparation.
• Dispense 25µl of each lyophilized controls in to its corresponding tube of 0.2 ml with micropipette.
• Store at -20°C in deep freeze no DDF-3. 

1.    Documentation

·       Log book of Biosafty Cabinet No. 7 PR-48

·       rRT PCR reagent Register PR-53

2.    Referance used to draw SOP

·       Manual On Intratypic differentiation of Polioviruses by Real time RT-PCR of CDC

·       Kit literature.