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
- 5 ul Tagmentation reaction
- 1-3 ul samples
- 1 ul 5X Tn5 buffer
- 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