Illumina DNA Prep with KAPA and Ampure SOP
Outlines below is the step by step SOP for the Illumina DNA Prep protocol with alternative reagents used in amplification and clean up
View red text where there are changes to the raw Illumina protocol here
Informative links
NOTE:
- ‘tube/well’ will be used throughout for clarity - the protocol can be complete in bulk (i.e 96 well plate) or with PCR tubes when testing or completed small batches
Before getting started…
- calculate your desired nDNA in the target volume per tube/well (C1V1 = C2V2)
- get your samples (i.e. from -80C) and thaw on the PCR cooler rack
- prepare your tubes/wells (pre labeled or a label sheet) wth the DNA diluted, if necessary, with Nuclease free DI (UltraPure DI)
Tagmentation
What you will need:
- Beaded Linked Transposome (BLT)
- Tagmentation Buffer 1 (TB1)
- PCR tubes or 96 well plate (with seal(s) such as Microseal ‘B’))
- Thermocycler, vortex
- pipettes, 1-10 ul
1) Thaw reagents Beaded Linked Transposome (BLT) and Tagmentation Buffer 1 (TB1) to room temperature
2) Create Tagmentation Master Mix (TMM) as the following:
n * 1.06 * 10 ul BLT (vortex vigorsouly before pipetting) +
n * 1.06 * 10 ul TB1
where n is the number of samples
3) Pipette your TMM 10x to mix
4) Place samples (PCR tubes or well) on thermocycler for TAG program
- review the link above for the Illumina TAG program, this is set on the thermocycler under folders ‘samgurr/Illumina_DNA_Prep’
Post Tagmentation Clean Up
What you will need:
- Tagmentation Stop Buffer (TSB)
- Tagmentation Wash Buffer (TWB)
- PCR tubes or 96 well plate (with seal(s) such as Microseal ‘B’))
- Thermocycler, vortex
- pipettes, 1-10 ul, 20 - 200 ul
1) Thaw Tagmentation Stop Buffer (TSB) and Tagmentation Wash Buffer (TWB) to room temperature
2) Remove the PCR tubes/plate from the thermocycler
3) Add 10 ul TSB to each sample. Pipette to mix & resuspend tthe beads (meaning the BLT beads in solution)
4) Seal the plate / close caps on PCR tubes
5) Place samples on the thermocycler and run the PTC program
- review the link above for the Illumina PTC program, this is set on the thermocycler under folders ‘samgurr/Illumina_DNA_Prep’
6) Remove from thermocycler
7) Place samples on a magnetic stand (we have one for plates and for PCR tubes/strips to accomodate bulk library prep and small batches). Wait ~ 3 minutes until liquid clears
8) Remove supernatant and discard
- note: use a chemical storage bottle for all liquid waste, properly labeled with % consituents in this library prep protocol
9) Wash 2X as the following:
a. Now with the supernatant discarded, remove sampels from the magnetic stand
b. Add 100 ul TWB slowly onto the beads. Pipette mix, also slowly, to resuspend the beads
c. place samples back onto the magnetic stand for ~3 minutes until liquid clears
d. Remove and discard the supernatant
9) After two washes completed, remove smaples from the magnetic stand and add 100 ul TWB slowly. Pipette mix, also slowly, to resuspend the beads
10) Seal the plate/ close tubes and place on the magnetic stand until Step 4 in Amplifying Tagmented DNA
Amplifying Tagmented DNA
What you will need:
- KAPA HiFi HotStart ReadyMix 2x (instead of Enhanced PCR Mix (EPM)!)
- Illumina Index Adapters (i.e. IDT, Nextera, as tubes or plates depending on kit size)
- Nuclease free DI water (UltraPure DI)
- PCR tubes or 96 well plate (with seal(s) such as Microseal ‘B’))
- Thermocycler, vortex
- pipettes, 1-10 ul, 20 - 200 ul
1) Thaw index adapters on 96 well or tube cooler rack depending on kit size
2) Place samples on magnetic stand for ~3 minutes until liquid clears and remove and discard the supernatant
3) Remove samples from the magnetic stand
4) Immediately Add 11.25 ul of KAPA HiFi HotStart ReadyMix into the beads. Pipette mix to resuspend
6) Seal plate/close tube caps and centrifuge for 3 seconds (benchtop centrifuge)
7) Add index adapters
- if tubes (24 samples kit) add 5ul of i5 and 5 ul of i7
- if a plate (96 well kit) add the contents of a single well (10 ul)
8) Pipette mix with a pipette set to 20 ul 10x to mix
9) Seal plate/close tube caps and centrifuge for 30 seconds (benchtop centrifuge)
10) Place samples on the thermocycler and run the modified annealing program by Nina/Harmony for Nextera library prep
- this is set on the thermocycler under folders ‘samgurr/Nextera_SOP’
Clean up
What you will need:
- Ampure XP beads (instead of Illumina Purification Beads!)
- 10mM tris pH 8 (instead of Resuspension Buffer (RSB)!)
- Freshly made 80% ethanol
- Nuclease free DI water (UltraPure DI)
- 3-4 sets of PCR tubes or 96 well plates (with seal(s) such as Microseal ‘B’))
- Thermocycler, vortex
- pipettes, 1-10 ul, 20 - 200 ul
Note: while the thermoccler is running, prepare fresh 80% ethanol to accomodate n * 400 ul +(1*n). You will require 400 ul per sample, an added 1 ul per sample to avoid pipette error.
1) Thaw Ampure XP beads to room temperature. Bring 10mM tris pH 8 to your bench (already stored at room temp).
2) Remove samples from the thermocycler
3) Centrifuge at 280 x g for 1 minute to collect beads at the bottom (I simgply used the benchtop centrifuge when working with PCR tubes and this worked fine)
4) Transfer 20 ul of the supernatant to new vessels (prelabled PCR tubes or same position on new 96 well plate). Discard previous tubes/plate
5) Add 12.49 ul of Ampure XP beads to each sample. Pipette mix 10x
8) Seal plate/close tube caps and incubate on the benchtop at room temperature for 5 minutes
9) Place samples on magnetic stand for 5 minutes until liquid clears. D
10) Remove and discard the supernatant careful to not discturb the beads
11) Wash 2X as the following:
a. With the samples on the magnetic stand, add 150 ul 80% ethanol without mixing
b. incubate for 30 seconds at room temperature
c. remove the supenatant without disturbing the beads
15) Use a 10-20 ul pipette to remove excess ethanol
17) Remove samples from the magnetic stand
18) Add 32 ul of 10mM tris pH 8 to each sample, beads should be resustpendided immediately (at least within 5 minutes to prevent overdrying). Pipette mix to resuspend beads.
19) Incubate at room temperature for 5 minutes
20) Place on magnetic stand for 2 minutes until liquid clears
21) Transfer 20 ul of supernatant to new vessles (prelabled PCR tubes or same position on new 96 well plate)