Arichard:Protocols/tn5059
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Tn5059 protocol (Supermutant) 2/25/2015[edit]
Prep[edit]
- Tn5059 was supplied at 25X, with transposons A and B in separate tubes. We are assuming that a 50/50 mix of A/B is 25X. We have both control transposons and T7 promoter transposons for Brandon's IVT project
- Follow the latest Nextera DNA sample prep user guide for tagmentation conditions and PCR. To avoid purification after tagmentation, use Qiagen Protease to inactivate the Tn5
- Dilute 25X Tn5 A/B mix to 5X in UV treated TE buffer with 50% glycerol before use. 1X Tn5 reaction in 5 ul has shown good fragment distribution for 6-8 ng of purified DNA (MDA product), but appears to over-fragment smaller template inputs
- This is similar to older Nextera
- Volumes below are per reaction. Make master mix with ~10% excess for multiple reactions
- Include both positive control (purified DNA with known concentration), NTC (No Template Control), and NEC (No Enzyme Control)
- UV treat tubes, buffer, and H2O before use
Protocol[edit]
- 5 ul Tagmentation reaction
- 3 ul template in H2O
- 1 ul 5X Tn5 buffer
- 1 ul 5X Tn5
- Incubate at 55 C for 5 minutes. Remove tubes to ice block
- Note: Brandon may have observed an increase in yield after incubating at room temperature for 1 hour before heating to 55 C
- Add 1 ul 1:100 diluted ProtQ
- Dilute 0.2 AU/ml Qiagen Protease 1:100 in H2O
- Incubate at 50 C for 10 minutes, followed by 70C for 20 minutes. Hold at 15 C
- Add 1 ul Exo- mix
- 0.1 ul 10 U/ul Exo-
- 0.4 ul 25 mM dNTPs
- 0.5 ul H2O
- Incubate at 37 C for 15 minutes, followed by 75 C for 20 minutes. Hold at 15 C
- Add 1 ul of i5 adapter (N/S5xx, containing index 2) and 1 ul i7 adapter (N7xx, containing index 1)
- Use low-plexity guidelines in Nextera user guide for small samples sizes
- Add 16 ul PCR mix
- 15 ul 2X PCR mix (I have used KAPA 2X SYBR Fast)
- 1 ul H2O
- PCR
- 72 C for 3 minutes
- 13 cycles of:
- 98 C for 10 seconds
- 63 C for 30 seconds
- 72 C for 3 minutes
- Hold at 15 C
- Purify using Ampure XP at 0.8:1 bead:sample volume ratio. Elute in 20 ul H2O
- Run 3 ul on TBE gel
- Purify again (same ratio) before sequencing (if low-weight fragments appear on gel)