Arichard:Protocols/tn5059: Difference between revisions

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>Andrew
(Created page with "==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 ...")
 
>Andrew
mNo edit summary
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* 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.
** This is similar to older Nextera


 
# 5 ul Tagmentation reaction
# 5 ul Tagmentation
## 1-3 ul samples
## 1-3 ul samples
## 1 ul 5X Tn5 buffer
## 1 ul 5X Tn5 buffer
## 1 ul 5X
## 1 ul 5X
# Incubate 5 minutes at 55 C. 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 50 C, followed by 20 minutes at 70 C.
# Add

Revision as of 19:37, 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
  1. 5 ul Tagmentation reaction
    1. 1-3 ul samples
    2. 1 ul 5X Tn5 buffer
    3. 1 ul 5X
  2. Incubate 5 minutes at 55 C. 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.
  1. Add 1 ul ProtQ. Incubate 10 minutes at 50 C, followed by 20 minutes at 70 C.
  2. Add