Arichard:Protocols/tn5059: Difference between revisions

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==Tn5059 protocol (Supermutant) 2/25/2015==
==Tn5059 protocol (Supermutant) 2/25/2015==


* 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.
===Prep===


* 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.
* 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


* 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.
* 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
** 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===


# 5 ul Tagmentation reaction
# 5 ul Tagmentation reaction
## 1-3 ul samples
## 3 ul template in H2O
## 1 ul 5X Tn5 buffer
## 1 ul 5X Tn5 buffer
## 1 ul 5X
## 1 ul 5X Tn5
# Incubate 5 minutes at 55 C. Remove tubes to ice block.
# 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 5 minutes before heating to 55 C.
## 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 ProtQ. Incubate 10 minutes at 50 C, followed by 20 minutes at 70 C.
# Add 1 ul 1:100 diluted ProtQ
# Add
## 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)

Latest revision as of 18:41, 26 February 2015

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]

  1. 5 ul Tagmentation reaction
    1. 3 ul template in H2O
    2. 1 ul 5X Tn5 buffer
    3. 1 ul 5X Tn5
  2. Incubate at 55 C for 5 minutes. Remove tubes to ice block
    1. Note: Brandon may have observed an increase in yield after incubating at room temperature for 1 hour before heating to 55 C
  3. Add 1 ul 1:100 diluted ProtQ
    1. Dilute 0.2 AU/ml Qiagen Protease 1:100 in H2O
  4. Incubate at 50 C for 10 minutes, followed by 70C for 20 minutes. Hold at 15 C
  5. Add 1 ul Exo- mix
    1. 0.1 ul 10 U/ul Exo-
    2. 0.4 ul 25 mM dNTPs
    3. 0.5 ul H2O
  6. Incubate at 37 C for 15 minutes, followed by 75 C for 20 minutes. Hold at 15 C
  7. Add 1 ul of i5 adapter (N/S5xx, containing index 2) and 1 ul i7 adapter (N7xx, containing index 1)
    1. Use low-plexity guidelines in Nextera user guide for small samples sizes
  8. Add 16 ul PCR mix
    1. 15 ul 2X PCR mix (I have used KAPA 2X SYBR Fast)
    2. 1 ul H2O
  9. PCR
    1. 72 C for 3 minutes
    2. 13 cycles of:
      1. 98 C for 10 seconds
      2. 63 C for 30 seconds
      3. 72 C for 3 minutes
    3. Hold at 15 C
  10. Purify using Ampure XP at 0.8:1 bead:sample volume ratio. Elute in 20 ul H2O
  11. Run 3 ul on TBE gel
  12. Purify again (same ratio) before sequencing (if low-weight fragments appear on gel)