Editing BioMicroCenter:Clustering

{{BioMicroCenter}}

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.