January April 20156 NGS Facility – Terms of Use

NGS FACILITY PROTOCOLS

LAST UPDATED: APRIL 2016

LIBRARY PREP (PRE-PCR) ROOM

  1. Room access
  • To get started, please go through induction process with one the NGS facility trainers:

Dominik Kaczorowski (ext.: 8283; email: )

Shalima Nair (ext.: 8386; email: )

Lee Marshall (ext.: 8279; email: )

Iva Nikolic (ext.: 5891;email:)

  • In addition, complete the online test (link), have the training sheet signed off by one of the trainers or Pavel, and obtain card access from Fiona Richards.
  • Before starting the procedure, we advise you to discuss it with more experienced users.
  • Please send your comments/thoughts to .
  1. Booking the workstations
  • There are two fully equipped workstations in the room (NGS_Workstation_1 and NGS_Workstation_2), each containing a full set of pipettes, a 96-well skirted magnetic stand, and a thermocycler with a temperature-sensitive lid.
  • A third workstation (NGS_PCR_Hood) contains in addition a portable UV hood and will be reserved mostly for single-cell/low-input library prep work.
  • Workstations can be booked in 5h intervals: from 9am-2pm, and from 2pm-7pm. Each time slot is sufficient for completing a part of the library prep protocol, which can then be continued the following day.

If you are an experienced user and you usually perform the whole protocol in a day, you are allowed to book both time slots (just be mindful of other users and their work).

  • If both NGS_Workstation_1 and NGS_Workstation_2 are occupied, it is possible to use NGS_PCR_Hood station for regular library preps. Please keep in mind that single-cell library prep protocol includes a cDNA amplification step and that, in theory, contamination of your samples is possible. Please follow the guidelines outlined below.
  • The workstations can be booked through the online booking system under the following names:

GMG_NGS_Workstation_1

GMG_NGS_Workstation_2

GMG_NGS_PCR_Hood

  • There is a $50 charge per 5h interval for booking any of the workstations.
  1. Terms of use
  • Samples allowed in this room are RNA and non-amplified DNA (fragmented and/or bisulfite converted).The only exception to this rule is a low-input/single-cell library prep protocol, which includes a low-cycle-number amplified cDNA.
  • Applications:

ALLOWED / NOT ALLOWED
RNA isolation in RNase-free fume hood / PCR amplification
rRNA depletion, polyA selection, DNase treatment / Any post-PCR work
RNA and DNA fragmentation
First-strand synthesis
Second-strand synthesis
Adenylation and end repair
Adapter ligation
Any pre-PCR cleanup steps
PCR setup
  1. Movement between pre-PCR and post-PCR rooms
  • Before starting the procedure, take a clean lab coat and label it clearly with your name. The lab coat should be worn inside the pre-PCR room only. It is critical not to move between the pre-PCR and post-PCR rooms in the same lab coat.
  • You are allowed to use the same gloves when moving from the pre-PCR room to the post-PCR room (taking care to avoid door handles), but not when moving in the opposite direction.
  • Keep all reagents such as ethanol, nuclease-free water, AMPure beads in the pre-PCR room. Take aliquots across to the post-PCR room and keep them there.
  • Do not transfer pipettes or other equipment between the rooms.
  • After finishing your work in the pre-PCR room, remove your lab coat.
  1. Terms for using the low-input library prep workstation (NGS_PCR_Hood)
  • Because the low-input library prep protocol includes a cDNA amplification step, it is critical to adhere to the outlined guidelines.
  • Familiarise yourself with the operation and limitations of the PCR cabinet (instructions are located on top of the cabinet).
  • Prior to use, wipe the interior of the PCR cabinet using DNA Away.
  • Keep all used tips in a closed waste container (e.g. empty tip box without the rack), and dispose of it immediately after use or when full.
  • Aliquot reagents OUTSIDE the PCR cabinet.
  • Change gloves regularly, especially when moving from the PCR cabinet to the general room area.
  • For the first, low cyclenumber cDNA amplification step common in low-input protocols, preform perform PCR in the designated PCR machine in the fume cabinet following the specific guidelines posted.post-PCR room (remember to take off your lab coat before leaving the clean room). After the PCR has finished, transfer the amplified mixture into a new, clean plate and continue working in the PCR cabinet. PLEASE NOTE: the final library amplification step MUST ALWAYS be performed in the post-PCR room ONLY!Move the plate back to the pre-PCR room and continue working with it only inside the PCR cabinet!
  • After finishing the library prep, remove the plate from the prepost-PCR room and store it in your own lab space.
  • With all consumables present in the PCR cabinet, switch on the UV lamp for 15-30min.
  • Remove all reagents and consumables from the PCR cabinet. Dispose of the empty ones and clean the ones for further storage with DNA Away.
  • Switch on the UV lamp in the empty PCR cabinet for 15-30 min, making sure to raise the internal glass shelf (instructions on top of the cabinet).
  • Wipe the interior of the PCR cabinet as well as all surrounding areas using DNA Away.
  1. Storage space
  • There is one fridge and one freezer in the pre-PCR room, and each group will be assigned their space according to the proportion of the facility users coming from that group.
  • Both the fridge and the freezer are to be used for storing reagents and working stocks only. This space should not be used for archiving.
  • Reagents should be kept in Tupperware containers, which should be clearly labelled with the group name and user name.
  1. Cleaning
  • Library prep room is cleaned on a weekly basis. Everyone using the facility will be included in the roster, displayed on the NGS confluence page.
  • Wipe all surfaces and pipettes using RNA Zap.
  • Check the waste, empty the bins if necessary, and replace the waste bags.
  • Tidy up the lab coats and remove the unlabelled ones.
  • Check the communal consumables and refill if necessary:

Tip boxes (10ul, 20ul, 200ul, 1000ul)

Nitrile gloves (small, medium, large)

Falcon tubes (50 ml and 15 ml)

Ethanol

Nuclease-free water

Kim wipes

RNA Zap

DNA Away

Waste bags

  • If any of the consumables is are running low, please contact GMG via email ()note it in the ordering sheet and forward it to GMG.
  1. Consumables
  • NGS facility stocks consumables such as gloves, tips, Falcon tubes, ethanol, and nuclease-free water. These consumables are communal and every facility user is responsible for checking the stocks and, if necessary, for their ordering (see above).
  • Other consumables including the plates, adhesive seals, and low-DNA binding eppendorf tubes should be supplied by the facility users.

POST-PCR ROOM

  1. Room access
  • There is no special access required for this room.
  1. Booking the workstations
  • There are two fully equipped workstations in the room (NGS_Post_PCR_1 and NGS_Post_PCR_2), each containing a set of pipettes, a 96-well skirted magnetic stand, and a thermocycler (note that ABI thermocyclers do not have temperature-sensitive lids).
  • Workstations can be booked in 2h intervals through the online booking system (GMG_NGS_Post_PCR_1 and GMG_NGS_Post_PCR_2).
  • There is a $20 charge per 2h interval for booking the workstations.
  • There is a Bio-Rad thermocycler available(with a temperature-sensitive lid), which can be booked separately (GMG_NGS_Post_PCR_Biorad). This machine will be reserved mostly for specific applications and long incubation steps.
  • The bioanalyzer should be booked separately through the online booking system.
  1. Terms of use
  • Samples allowed in this room are PCR-amplified samples.
  • Applications:

ALLOWED / NOT ALLOWED
PCR amplification of libraries / None
Post-PCR cleanup of libraries
Hybridization of PCR-amplified libraries
Bioanalyzer assays:
RNA – nano and pico kits
DNA – DNA1000 and HS kits
  1. Storage space
  • There is one fridge and one freezer in theroom, and each group will be assigned their space according to the proportion of the facility users coming from that group.
  • Both the fridge and the freezer are to be used for storing reagents and working stocks only. This space should not be used for archiving.
  • Reagents should be kept in Tupperware containers, which should be clearly labelled with the group name and user name.
  1. Cleaning
  • A weekly cleaning routing (see section Library prep room/cleaning) includes also the post-PCR room.
  • Wipe all surfaces and pipettes using DNA Away.
  • Check the waste, empty the bins if necessary, and replace the bin waste bags.
  • Tidy up the lab coats and remove the unlabelled ones.
  • Check the communal consumables and refill if necessary:

Tip boxes (10ul, 20ul, 200ul, 1000ul)

Nitrile gloves (small, medium, large)

Falcon tubes (50 ml and 15 ml)

Kim wipes

DNA Away

Waste bags

  • If any of the consumables is running low, note it in the ordering sheet and forward it to GMG.
  1. Consumables
  • NGS facility stocks consumables such as gloves, tips, and Falcon tubes. These consumables are communal and every facility user is responsible for checking the stocks and, if necessary, for their ordering (see above).

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