AlanFung:Protocol/Genome Analyzer Reagents Prep
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Preparing Reagents for the Genome Analyzer[edit]
Genome analyzer Reagent Compartment & Reagent Position[edit]
- Position of each reagent in the Genome Analyzer
NOTE:LABEL ALL REAGENT CONTAINERS WITH THE APPROPRIATE NUMBER Position Reagent Volume Container 1 #Incorporation Mix Varies w/ cycle # 50ml amber tube 2 DI H2O 250ml bottle 3 #Scanning Mix 50ml 50ml tube 4 #High Salt Buffer 250ml bottle 5 #Incorporation buffer 150ml 250ml bottle 6 #Cleavage Mix 50ml 50ml tube 7 #Cleavage Buffer 70ml 250ml bottle
Scanning Mix[edit]
Required Materials *Scanning Reagent *Scanning Buffer *Filter Unit *Scanning Mix Tube
- Add the Scanning Reagent vial to the Scanning Buffer on ice
- Rinse the empty vial into the Scanning Buffer three times with 1 ml Scanning Buffer
- Invert the Scanning Buffer several times to mix
- Leave on ice for 10 minutes
- Invert several times to mix. Check to ensure that the solids are completely dissolved
- Filter the Scanning Mix with the provided Scanning Mix Filter Unit
- Pour 50 ml of the Scanning Mix into the 50 ml Scanning Mix Tube
*If you are running more than 26 cycles on the Genome Analyzer, you must top off the Scanning Mix at the end of the first day of the sequencing run. The tube should be approximately one-third empty after running for six or seven hours
- Record the weight of the reagent in the lab tracking worksheet
- Leave the remaining solution in the Filter Unit at 4C for later cycles
- Keep the Scanning Mix on ice until ready to load
Incorporation Mix[edit]
Required Materials *The volumes of the dNTPs in the various kits are specific to that kit *Do not mix and match dNTP vials form different cycle number kits *dNTPs: ff-dATP, ff-dCTP, ff-dGTP, ff-dTTP 2 each for the 36-cycle kit 1 each for the 18-cycle and 26-cycle kits *10X Incorporation Buffer v2.0 (5 ml) *dNTP Mix Diluent *SBS Polymerase 2 for the 36-cycle kit 1 for the 18-cycle and 26-cycle kits *Magnesium *Plastic Syringe *0.22 μm Cellulose Acetate Syringe Filter *50 ml Amber Tube
- The volumes of dNTP Diluent, 10X Incorporation Buffer, and Magnesium vary depending on the sequencing kit you are using
- Refer to the table below for correct volumes
Reagents 18-Cycle 26-Cycle 36-Cycle dNTP Diluent 21ml 29ml 43ml 10X Incorporation Bufferv2.0 2.5ml 3.5ml 5ml Magnesium 150ul 210ul 300ul
- Transfer the correct volume of dNTP Diluent to a 50 ml tube
- Refer to the table of volumes for the sequencing kit you are using
- Add the correct volume of 10X Incorporation Buffer v2.0
- Refer to the table of volumes for the sequencing kit you are using.
- Vortex for 10 seconds.
*NOTE Save the remaining 10X Buffer for the Incorporation Buffer
- Pipette the contents of every ff-dNTP vial into the Incorporation Mix
- Rinse each of the ff-dNTP tubes into the dNTP mix with 1 ml of Incorporation Mix
- Invert several times to mix
- Filter the dNTP mix through a 0.22 μm membrane filter
- Transfer the mix into the supplied 50 ml amber tube labelled “dNTP Mix Tube.”
- Place the dNTP mix on ice.
- Remove the Magnesium solution from ice and allow it to thaw at room temperature for 10 minutes.
- Vortex to mix.
- Centrifuge to 10,000 xg for 2 minutes at 22°C.
- Ensure that the vial contains no undissolved material or ice.
- Add the correct volume of Magnesium solution to the dNTP mix on ice.
- Refer to the table of volumes for the sequencing kit you are using.
- Invert several times to mix and return to ice.
- Centrifuge the SBS Polymerase to 10,000 xg for 1 minute at 22°C.
*NOTE Two vials of SBS Polymerase are used in the 36-Cycle Kit. Only one vial of SBS Polymerase is used in the 18-Cycle and 26-Cycle Kits
- Add the contents of each polymerase vial into the dNTP mix on ice.
- Rinse each empty vial into the dNTPs mix three times with 1 ml dNTP mix
- Record the weight of the reagent in the lab tracking worksheet.
- Invert the dNTP mix several times to mix and immediately return to ice.
- Keep on ice until ready to load.
Incorporation Buffer[edit]
Required Materials *10X Incorporation Buffer v2.0 *Incorporation Buffer Diluent
- Add 15ml of 10X Incorporation Buffer v2.0 to the 150ml bottle of Incorporation Buffer Diluent
- Record the weight of the reagent in the lab tracking worksheet
High Salt Buffer[edit]
- Invert the High Salt Buffer several times to mix it before loading it on the Genome Analyzer
- Record the weight of the reagent in the lab tracking worksheet
1X Cleavage Buffer[edit]
Required Materials 10X Cleavage Buffer v2.0 (10ml) Cleavage Buffer Diluent (63ml)
- Add 7ml of 10X Cleavage buffer v.2.0 to the 150ml bottle of Cleavage Buffer Diluent
- Invert several times to mix
- Record the weight of the reagent in the lab tracking worksheet
- Store in ice until ready to use
Cleavage Mix[edit]
Required Materials Cleavage Reagent (5ml) Cleavage Mix Diluent (40ml) Cleavage Mix Additive 10X Cleavage buffer v2.0 (5ml left over from 1X cleavage Buffer preparation) Plastic Syringe 0.22um Cellulose Acetate Syringe Filter 50ml Tube (User-Supplied)
- Place the Cleavage Reagent in a 37°C circulating water bath.
- When the reagent is completely thawed, invert it several times to mix
- Transfer it back to ice immediately
- Add 5 ml of 10X Cleavage Buffer v2.0 to the tube of Cleavage Mix Diluent
- Add 5 ml of the thawed Cleavage Reagent to the tube of Cleavage Mix Diluent
- Invert several times to mix.
- Add the Cleavage Mix Additive to the Cleavage Mix Diluent tube. This solution is now called the Cleavage Mix. Rinse the empty additive tube into the Cleavage Mix three times with 1 ml of Cleavage Mix.
- Invert several times to mix. Return the tube to ice and leave it until the solids are completely dissolved
- Filter the Cleavage Mix into a new 50 ml tube using the syringe and 0.22 μm cellulose acetate syringe filter provided in the kit
- Label the tube “Cleavage Mix.”
- Record the weight of the reagent in the lab tracking worksheet
- Return the filtered mix to ice immediately
- Discard your gloves and replace with a new pair