Repeat methyltransferase of the Y-adapters[edit]
Purpose[edit]
- Methylate all cytosine residues within the double-stranded dinucleotide recognition sequence 5'...CG...3'. So that all cytosine at the CG site will not be converted during bisulfite conversion.
Improvements made from 2010-2-15 experiment[edit]
- Kun suggests that we use 2-5X more enzyme to ensure the completion of the reactions
- Also the volume of undiluted enzyme should be no more than 1/10 of the total reaction volume, for glycerol in the enzyme tends to inhibit the activity of the enzyme
- Use 1ul of 400U/ml enzyme for 1ug for adapter
- Double the amount of adapter to 2ug
- Increase incubation time to 2 hours, allow methyltransferase reaction to go to completion
Protocol[edit]
NEBuffer (10X) |
2.5 |
2.5
|
Sam (20X) |
1.25 |
1.25
|
2ug PE_b_Met |
8.66 |
N/A
|
2ug PE_t_Met |
N/A |
7.46
|
M.Sssl (4U/ul) |
2 |
2
|
ddH2O |
10.59 |
11.79
|
Total |
25 |
25
|
|
- Incubate at 37C for 2hr , 65C for 20 mins
Removal of biotin strand from adapter[edit]
Purpose[edit]
- Removal of the strand with biotin group using magnetic beads, so that we can obtain the top and bottom portion of the PE_b_Met and PE_t_Met adapter.
- Use 1mg of Dynabeads (100ul) to 10ug of ds DNA
Material[edit]
- Dynabeads M-280 Streptavidin_Invitrogen_Cat. no. 112.05D (10mg, 6-7*10^8 Dynabeads per ml)
- 2x Binding and Washing Buffer (B&W Buffer)
-[10mM Tris-HCL (pH 7.5)]
-[1mM EDTA]
-[2M NACL]
Prepare 10ml B&W Buffer
|
ul
|
5M NACL |
4000
|
0.5M EDTA |
20
|
1M Tris-HCL |
100
|
ddH2O |
5880
|
Total |
10000
|
|
Prepare Streptavidin Dynabeads[edit]
- Take 100ul Dynabeads per reaction, place the tube on magnet and remove the supernatant when the solution becomes clear ~2 mins
- Remove the tube from magnet
- Add twice the volume of the beads of 1x binding buffer along the the inside of the tube where the beads are collected and resuspend
- Place the tube on magnet and remove the liquid when the solution becomes clear
- Wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA)
- Resuspend the beads to 5ug/ul, 200ul of 2X B&W buffer
Immobilization of Nucleic Acids[edit]
- Add the 25ul primer to 25ul Dynabeads, incubate at RT for 15 mins in the Thermal Mixer.
Binding to magnet and washing[edit]
- Seperate the biotinylated primer with a magnet for 3 mins
- Remove the liquid from the beads with a magnet.
- Wash the coated beads 3 times with 1XB&W buffer
Denature using NaOH[edit]
- After the final wash, rinse the beads with 50ul 0.1M NaOH, incubate at RT for 1min.
Neutralize Product[edit]
- Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted primer) to 70ul 1M Tris-HCl, pH 7.5.
- Purify the 120ul neutralized primer by ethanol precipitation with glyco blue, resuspend with ddh2o
- Add 12 ul 3M sodium acetate and 300ul 100% ethanol with 1ul glycoblue
- Freeze in -80C for 20 mins
- Spin at 10,000rpm for 20min at 4C
- discard supernatant
- Add 500ul 75% etoh spin at 10,000 for 5 min at 4C
- Discard supernatant
- let dry in hood ~10min
- Add 20ul to resolve adapter