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==='''cluster generation protocols and recipes'''=== *check the cluster station recipes table 2 for your application *check table 5 to determine which reagents you need to prepare and load for the recipes you are using ==='''preparing DNA template for cluster generation'''=== *HP3 *HT1 *Tris-CL 10mM pH 8.5 ===template mix=== *denature the template dna with hp3 to a final dna concentration of 1nM *combine the following volume of template dna , tris-cl and hp3 10nM template(2uL) tris-cl 10mM ph 7.5 (17ul) hp3 (1uL) *vortex briefly to mix the template solution *pulse centrifuge the solution *incubate for 5' at rt to denature the template into single strands *place the template on ice until you are ready to proceed to final dilution ===dilute denature dna=== *dilute the denatured dna with pre chilled ht1 to total volume of 1000ul and dispense into a strip tube. *to reach the desired final concentration for the hybridization step dilute denatured dna as follows *invert several times to mix the template solution *pulse centrifuge the solution *dispense 120ul of illumina control dna library into tube 5 of .2ml strip *label the tube strip B *set aside on ice until you are ready to load it onto the cluster station ===template mix=== ==='''Performing linearization, blocking , and primer hybridization'''=== ===wash the cluster station=== *select file|open recipe *open the standard protocol recipe and click run *install the washing bridge and load containers filled with water in the following positions *single-read recipe-load position 3,4,7,12,15,17 *paired-end recipe-load position 3,4,7,12,14,17 *click ok to proceed and follow the prompts when the wash is complete *remove water from the following positions *single-read recipe-load position 3,4,7,12,15,17 *paired-end recipe-load position 3,4,7,12,14,17 *click ok and follow the prompts when the air gap has been primed ===loading reagents=== best practices always remove and replace reagents one tube at a time wear gloves at all times do not touch reagents with bare hands connect the tubes by holding the caps stationary while you twist the tubes into place to prevent crimping and twisting of the lines ===perform linearization blocking and primer hybridization=== *load reagents in the following positions as indicated *single-read recipe-load position 3,4,7,12,15,17 *paired-end recipe-load position 3,4,7,12,14,17 *click ok to resume the recipe and follow the prompt to proceed to linearization blocking and primer hybridization *load flow cell and attach the amplification manifold *click ok to proceed and follow the prompt to check for proper flow *as the process starts check for correct fluid flow through all eight lanes and the lines of the amplification manifold when the flow is regular in all lanes proceed with the protocol *click ok to proceed *wait for the message to let you know that the flow cell is ready for sequencing *remove the flow cell from the cluster station DO NOT STORE THE FLOW CELL AT THIS POINT FOR LONG PERIODS OF TIME. SEQUENCING MUST BE PERFORMED ON THE FLOW CELL WITHIN 4 HOURS *Measure the reagent volumes and record them on the lab tracking form. compare to the table provided ===wash the cluster station=== *click ok to resume the recipe *attach the washing bridge and load containers filled with water in the following positions: *single-read recipe-load position 3,4,7,12,15,17 *paired-end recipe-load position 3,4,7,12,14,17 *click ok to proceed, wait till a message appears to let you know that the cycle is complete A WEEKLY DECON WASH IS REQUIRED Using the recipe DECON_WASH_ALL_Linesv3.0xml. *The decon solution consists of 5% decon in water. all lanes are washed once with decon solution followed by two washes with water
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