Chris:LabNotes/sci-Methyl Seq/Calendar/2017/2017-5-17: Difference between revisions
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>Cjwei (Created page with "=Test Nuclei Loss= ==Background== *We've noticed that we are getting ~75% loss of nuclei with every centrifugation during our adapter ligation. *Here are some notes from other...") |
>Cjwei No edit summary |
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= | =sci-Methyl Seq Barcode 1 Design v2= | ||
==Background== | ==Background== | ||
* | *Previously, I've designed Adapter 1 with a CG sticky end that would anneal directly onto MspI digested DNA. However, as previous tests have shown, this approach doesn't work because of the high amounts of adapter-adapter and template-template annealing that goes on. | ||
* | *Dr. Zhang then suggested that we look into using end-repair/dA-tailing prior to adapter ligation. This will ensure that the majority of ligated product are template+adapter. Consequently, we want to make a slight edit to the Adapter 1 test design to include a T-overhang instead of current CG overhang | ||
==Barcode 1 Adapter Design (v2)== | |||
*Based on what we know from dA-tailing, an A will be added to the 3' end of blunt-ended DNA. Consequently, the DNA fragment after end-repair/dA-tailing would be: | |||
5' -------------------A 3' | |||
* | 3' A------------------- 5' | ||
*Consequently, our Adapter 1 v2 design should be as follows: (remember we want to create a dsDNA adapter for ligation) | |||
5' /5Phos/------------------- 3' | |||
*Below are the | 3' T------------------- 5' | ||
*Below are the sequences we want to order from IDT: | |||
Adpt1_v2 5' /5Phos/TTAGAGGTGGTCCCTCCTACCCGGCGTTT 3' | |||
Adpt1_v2_comp 3' TAATCTCCACCAGGGAGGATGGGCCGCAAA 5' |
Latest revision as of 19:34, 17 May 2017
sci-Methyl Seq Barcode 1 Design v2[edit]
Background[edit]
- Previously, I've designed Adapter 1 with a CG sticky end that would anneal directly onto MspI digested DNA. However, as previous tests have shown, this approach doesn't work because of the high amounts of adapter-adapter and template-template annealing that goes on.
- Dr. Zhang then suggested that we look into using end-repair/dA-tailing prior to adapter ligation. This will ensure that the majority of ligated product are template+adapter. Consequently, we want to make a slight edit to the Adapter 1 test design to include a T-overhang instead of current CG overhang
Barcode 1 Adapter Design (v2)[edit]
- Based on what we know from dA-tailing, an A will be added to the 3' end of blunt-ended DNA. Consequently, the DNA fragment after end-repair/dA-tailing would be:
5' -------------------A 3' 3' A------------------- 5'
- Consequently, our Adapter 1 v2 design should be as follows: (remember we want to create a dsDNA adapter for ligation)
5' /5Phos/------------------- 3' 3' T------------------- 5'
- Below are the sequences we want to order from IDT:
Adpt1_v2 5' /5Phos/TTAGAGGTGGTCCCTCCTACCCGGCGTTT 3' Adpt1_v2_comp 3' TAATCTCCACCAGGGAGGATGGGCCGCAAA 5'