Continuation of Library Construction[edit]
[[1]]
MET_Y_adapter Purification[edit]
- Perform ethanol precipitation to purify adapter
- add 10ul 3M NaoAC and 250ul 100% EtoH and 1ul glycoblue mix and freeze in -80C for 20M
- spin at 10,000rpm for 20 mins at 4C
- discard supernatant
- Add 500ul of 75% etoh and spin at 10,000rpm at 4C
- discard supernatant and let dry in hood for 10mins
- resolve in 100ul ddh2o
TBE gel verification[edit]
File:ZhangLab 2 2010-02-22 14hr 08min.jpg
- Band with very large size bp showed up adapter should be in there, move on with adapter ligation
Quantify using qubit[edit]
- Mix 1ul dye with 199ul buffer
- Mix 2ul adapter with 98ul diluted dye
- mix and incubate for 2 mins
Results: 4.24ng/ul *100ul = 424ng
Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences[edit]
- Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
|
ul
|
A-tailed input DNA |
50
|
100ng MET_Y_adapter(4.24ng/ul) |
23
|
5X Quick Ligase Buffer |
20
|
Quick T4 DNA Ligase |
3
|
ddh2o |
5
|
|
Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 30ul EB.
- Add 5X Volume PB w/ sample mix
- spin and discard at 14,000rpm for 1min
- add 750ul wash buffer
- spin and discard x2
- Add 32ul eb buffer wait for 5 mins and spin to collect tube
- Aliquot 28ul for CT conversion and 2ul for qPCR with PhiX
Perform PCR to the bisulfite converted DNA using Phusion/iProof (Stop @ Plateau)[edit]
Prepare CT Conversion Reagent[edit]
- CT Conversion Reagent is light sensitive, so minize its exposure to light
- Add 790ul M-solubilization buffer and 300ul M-Dilution Buffer to a tube of CT Conversion Reagent
- Mix by frequent vortexing at RT for 10m
- Mix 160ul of M-Reaction Buffer and mix an additional 1m
I suspect the adapter that act as carrier will be lost during purification for minelute only binds dsDNA 70bp and above and hence need to add in tRNA as carrier for bisulfite conversion
Prepare Carrier[edit]
To get 500ng tRNA
Take 2.463ul tRNA from (203ng/ul tRNA)
Prepare conversion[edit]
|
5ng+carrier |
10ng + Carrier
|
Volume from DNA |
28 |
28
|
Volume from tRNA |
2.463 |
2.463
|
|
Bisulfite conversion of input DNA with PE_Y_Met adapter[edit]
- Add sample with carrier to 130ul of CT conversion reagent solution in a low bind .5ml tube
- Vortex to mix
- Pulse centrifuge
- Perform
*98C for 8m
*64C for 3.5hr
*4C hold
- Add 600ul of M binding buffer into a column assembly
- Load sample(s) to the column
- Close the cap and mix by inverting the column several times
- Centrifuge at >10,000g for 30sec
- Discard the flow through
- Add 100ul of M-Wash Buffer to the column
- Centrifuge at full speed for 30sec
- Add 200ul of M-Desulphonation buffer to the column and let stand at RT for 20m
- Centrifuge for 30sec
- Add 100ul of M-Wash buffer to the column and repeat centrifuge
- Place the column into a 1.5ml low bind tube
- Add 11ul of M-elution buffer directly to the column matrix (Volume can be adjusted, depending on the requirements)
- Centrifuge for 30sec to elute the DNA
PCR Amplification of input DNA library[edit]
|
1 rxn |
Prepare
|
DNA |
20 |
|
H2O |
0.8 |
1.76
|
PCR_F (10uM) |
2 |
4.4
|
PCR_R (10uM) |
2 |
4.4
|
SYBR 50X |
0.2 |
0.44
|
2X Fusion Enzyme |
25 |
55
|
Total |
50 |
110
|
|
- Add 30ulmix to 20ul library
perform
- 98C 30sec
- 98C 10 sec
- 64C 20 sec
- 72C 20sec
- plate read
- line 2 4 more times
- 98C 10sec
- 72C 20sec
- plate read
- goto line 7 5 more times
- 72C 5 min
- 15C hold
TBE Gel quantification[edit]
File:ZhangLab 2 2010-02-24 14hr 48min.jpg
- Expected band showing up as a smear around 500BP for input DNA is a smear around 400bp when ligated to adapter(~119bp)
- No visible band shows up at 500bp
- Could be due to lose of DNA during purification using minelute