Preparing Reagents for the Cluster Station
Denaturing and preparing samples:
- Label two rows of 1.5mL microcentrifuge tubes 1 through 8, one for denaturation and one for final dilution (this one is the pre-chilled hyb buffer)
- Dilute samples to 10nM using Illumina Dilution excel worksheet (do this into 1st row of labeled tubes, don’t need to dilute Phix)
- Aliquot 996uL of Hybridization buffer into 8 1.5mL microcentrifuge tubes (2nd row of labeled tubes) place these on ice (amount of Hybridization buffer will change if doing over 4pM)
- Make master mix of EB and 2 N NaOh, 162uL EB + 9uL NaOH (total of 171uL) vortex and spin (the amount of EB will change if doing over 4pM)
- Aliquot 19uL of EB/NaOH master mix into 8 1.5mL microcentrifuge tubes (2nd row of labeled tubes). Then add 1uL of each sample (including Phix). These amounts will change if doing over 4pM. Vortex, spin, and place on ice.
- Add 4uL of each denatured sample to the tubes containing the pre-chilled Hybridization buffer (this amount will change if doing over 4pM). Vortex and spin. Keep these tubes on ice until you are ready to load stip B onto the cluster station.
- Take out -20 cluster box. Take everything out. Put the enzymes and nucleotides on ice and leave TE and cluster buff out on the bench to defrost.
- Start making the Amp, Lin, Block reagents:
Label 6 50mL tubes Reagents #1, #9, #10, #11, #12, and Amplification Pre-mix
Label two 15mL tubes Reagent #3 and Blocking Buffer1X
Label three 1.5mL screw caps Reagent #5, #14, and #15
Also label a 1.5mL microcentrifuge tube "E"
- Reagent #3 (Linearization buffer):
o Add 1518uL (or 759uL x 2) of dH2O to the tube of Sodium Periodate o Vortex until dissolved o In a 15mL conical tube, add 60uL 1.0 M Tris to 1500uL formamide o Mix thoroughly o Transfer 1437uL (or 718.5uL x 2) of the sodium perodate solution into the 15mL conical tube of formamide/tris solution. o Mix thoroughly. o Add 2.3uL of 3APL to the solution in the 15mL conical tube (NOTE: 3APL is a viscous solution. Wipe and excess from the outside of the tip before adding it.) o Mix thoroughly
o Set aside until you are ready to load the reagents on the cluster station.
- Reagent #15 (Blocking Buffer 1X): o Dilute 300uL Blocking buffer 1 to 1x concentration with 2700uL deionized water o Transfer 1400uL of the 1x blocking buffer into a 1.5mL screw-cap tube o Set aside until ready to load reagents on the cluster station - Reagent #5 (Blocking Mix): o Prepare the blocking mix by mixing the following on ice: 1510.2uL (or 755.1uL x 2) of Blocking Buffer 1X 30.1uL of 130uM ddNTPs 19.7uL of Terminal Transferase the total volume is 1560uL o Set aside on ice until ready to load it onto the cluster station - Reagent #9 (Formamide): o Transfer 15mL of formamide into the tube - Reagent #10 (Wash buffer) o Transfer 10mL of wash buffer into the tube - Reagent #12 (Storage buffer) o Transfer 5mL of storage buffer into the tube - Reagent #14 (Deionized Water) o Transfer 1.5mL into the tube - Reagent #11 (Amplification Pre-Mix) o To prepare this mix the following into a 50mL tube: 15mL Water 3mL Cluster buffer 12mL Betaine 5M The total volume should be 30mL o If the room temp is high this mix might be cloudy, you can put it on ice to avoid this o Filter this mix with a 0.2um cellulose acetate syringe filter. o Transfer 12mL of the Amplification mix to the 50mL tune labeled #11 and leave the rest for use in other reagents.
- Tube Strip E (initial extension mix) o Initial extension mix using Taq polymerase: o Prepare this mix by mixing the following on ice in a 1.5mL epindorph tube: 975uL of Amplificatn pre-mix 20uL of 10mM dNTPs 5uL of Taq DNA polymerase The total volume should be 1000uL o Place this on ice until tube strip D is on the cluster station
- Reagent #1 (Amplification mix with Bst DNA Polymerase) o Mix the following in the 50mL tube: 12mL Amplification Pre mix 240uL of 10mM dNTPs 120uL of Bst DNA Polymerase The total volume should be 12.36mL o Set this tube aside on ice until you are ready to load it on the cluster station - Tube Strip A:
o Aliquot 140uL pf Hybridization buffer into each tube of a tube strip
- Tube Strip B: o Strip B is 120uL of each sample in the tubes of a tube strip (don’t do this until strip A is on the cluster station)
- Tube Strip C:
o Aliquot 100uL of wash buffer into each tube of a tube strip (don’t do this until B is on the cluster station
- Tube Strip D: o Add 100uL of the amplification mix into each tube of a tube strip, don’t do this until C is on the cluster station - Tube Strip E: o Use initial extension mix made above, on ice, aliquot 120uL into each tube, don’t do this until D is on the cluster station.