AlanFung:LabNotes/CTCF/2010-2-16

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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