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 | * 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 | * 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 | * 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 | |||
# 5 ul Tagmentation reaction | # 5 ul Tagmentation reaction | ||
## | ## 3 ul template in H2O | ||
## 1 ul 5X Tn5 buffer | ## 1 ul 5X Tn5 buffer | ||
## 1 ul 5X | ## 1 ul 5X Tn5 | ||
# Incubate | # 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 | |||
# Add 1 ul ProtQ. Incubate 10 minutes at | # 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) |
Revision as of 19:54, 25 February 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
- 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
- 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 5 minutes 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)