HS read 2 run
Материал из Zbio
[править] Preparing ICR for Read 2
[править] Thaw Reagents for 101cycles
- [ ] leave LRP (Long Read DNA Polymerase) at -20°C until mixing to ICR
- [ ] replace two tubes of LFN (nucleotides) from -20°C to 4°C and thaw overnight
- [ ] invert each tube several times and ensure there are completely thawed.
- [ ] use ICR directly from 4°C
[править] Prepare ICR for 101 cycles
- [ ] add the contents of two tubes of LFN to the bottle containing 47 ml of ICR.
- [ ] rinse each tube of LFN with ICR to ensure that all LFN is transferred
- [ ] aliquot 1.1 ml of LRP into the ICR/LFN mix
- [ ] cap the bottle and invert several times to mix
- [ ] set the mix on ice until loading reagents onto the HiSeq.
[править] Preparing PE- Reagents
»the following reagents are used for Read 2 (resynthesis) and are provided in HiSeq Read 2 Cluster Resynthesis Kit, which is box 2 of the HiSeq Paired-End Cluster Generation Kit.
»take following reagents from - 20°C storage and thaw them at RT or in a beaker with RT water:
• HT2 (Wash Buffer)
• HP3 (2 N NaOH)
• AT2 (100% Formamide)
• APM2 (AMX2 Premix)
• AMX2 (Amplification Mix 2)
• BMX (Blocking Mix)
• LMX2 (Linearization Mix 2)
• RMX (Resynthesis Mix)
• HP2 (Sequencing Primer Mix 2) (for standard paired-end runs)
[править] Prepare Reagents
Prepare RMX, LMX2, BMX, and AMX2 :
- [ ] invert each thawed tube to mix the reagent
- [ ] put reagents on ice until ready for loading onto the HiSeq.
Prepare APM2, AT2, and HT2 :
- [ ] invert each thawed tube to mix the reagent.
- [ ] keep reagents at room temperature until loading onto the HiSeq.
Prepare HP3 Dilute HP3 (2 N NaOH) to 0.1 N NaOH prior to loading it onto the HiSeq.
- [ ] vortex the thawed tube of HP3 and then pulse spin down.
- [ ] transfer 2.85 ml of PW1 (provided in the HiSeq Sequencing Kit) into a 15 ml falcon tube and add 150 μl of HP3
- [ ] vortex and spin down
- [ ] keep the NaOH mix on RT until loading onto the HiSeq
Prepare HP2 (Standard PE runs)
- [ ] vortex both thawed tubes and spin down
- [ ] keep the primermix on RT until loading onto the HiSeq
[править] Loading ICR for Read 2 onto the HiSeq
» before starting Read 2 load fresh ICR
- [ ] record the weight of ICR on the lab tracking form
- [ ] open the reagent door, raise the sippers for the reagent rack and replace (position 1) the fresh ICR with existing mix
- [ ] change the funnel cap to the fresh bottle of ICR mix
- [ ] lower the sipper for the reagent rack into the reagent bottles and make sure they are centered inside the bottles
[править] Loading of PE Reagents onto the HiSeq
» load the PE-reagents onto the PE -reagent rack before starting of read 2-resynthesis
- [ ] record the weight of each reagent on the lab tracking form
- [ ] make sure the PE rack is not in use for Read 2 resynthesis or Index Read preparation on the other flow cell.
- [ ] raise the sippers for the PE reagent rack, remove the rack and put in the PE-reagent tubes
10 RMX Resynthesis Mix
11 LMX2 Linearization Mix 2
12 BMX Blocking Mix
13 AMX2 Amplification Mix 2
14 APM2 AMX2 Premix
15 AT2 100% Formamide
16 HP7 Sequencing Primer Mix 2
18 HP3 Denaturation Solution
19 HT2 Wash Buffer
- [ ] lower the sippers into the paired-end reagent tubes and make sure that the sippers are centred inside the tubes
- [ ] close the reagent door and press next to resume the run