Noi/NOTES/2014-12-11: Difference between revisions
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>Noi (Created page with "= RRBS library prep, mouse Mouse hippocampus gDNA (Zhou's lab) = '''Back to calendar'''<br> * 2014-12-01: Received 9 mouse hippocamplus gDNA fro...") |
>Noi mNo edit summary |
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== 2) End-repair/dA-tailing == | == 2) End-repair/dA-tailing == | ||
* After MspI, I took 2ul of MspI-digested DNA for gel verification (equal to DNA ~33.33ng). Total volume left is 28ul | |||
* Mix 13ul of 5U/ul Klenow exo- (Thermoscientific) with 13ul of 20:2:2mM dAdCdG | |||
* Add 2ul of Klenow exo-/dAdCdG mix to each tube. Total volume is 30ul | |||
:- Spin down the tubes | |||
:- Mix by gentle pulse-vortexing on PCR rack 10x | |||
:- Spin down the tubes | |||
:- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) | |||
:- 75C, 10min --> hold at 4C (heat lid on) | |||
:- Perform AMPure bead purification with 2x volume AMPure beads | |||
==== <u>AMPure bead purification</u> ==== | |||
:- Add 60ul AMPure bead. Mix by pipetting 10x | |||
:- Wait for 15min | |||
:- Transfer to sit on magnet for 5min | |||
:- Wash twice with 200ul freshly prepared 80% EtOH | |||
::- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x | |||
:- Remove all 80% EtOH | |||
:- Dry the bead for 15min | |||
:- Resuspend with 20 EB Buffer <u>''(keep the beads in the tubes!)''</u> | |||
== Methylated adaptor ligation == | |||
{| {{table}} border = 1 | |||
| align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''11rxn Mix''' | |||
|- | |||
| dA-tailed DNA||20.00||0.00 | |||
|- | |||
| 10x Ligation Buffer||3.00||33.00 | |||
|- | |||
| T4 DNA Ligase (6000U/ul)||1.00||11.00 | |||
|- | |||
| TruSeq Adaptor (15uM)||1.50||0.00 | |||
|- | |||
| H2O||4.50||49.50 | |||
|- | |||
| Total||30.00|| | |||
|} | |||
:- Add 1.5ul of 15uM (non-diluted adaptor) TruSeq adaptor to each tube | |||
:{| {{table}} class = wikitable | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Truseq Index''' | |||
|- | |||
| HC-C1||1 | |||
|- | |||
| HC-C2||2 | |||
|- | |||
| HC-C3||3 | |||
|- | |||
| HC-C4||4 | |||
|- | |||
| HC-H1||5 | |||
|- | |||
| HC-H2||6 | |||
|- | |||
| HC-H3||7 | |||
|- | |||
| HC-H4||8 | |||
|- | |||
| HC-H5||9 | |||
|- | |||
| NTC||10 | |||
|} | |||
* Final conc. of adaptor in 30ul lgation reaction is 750nM | |||
:- Add 8.5ul of ligation reaction mix with adaptors | |||
:- Spin down the tube | |||
:- Mix by gentle pulse-vortexing on PCR rack 10x | |||
:- Spin down the tube | |||
:- 16C, 24h (no heat lid) | |||
:- Mix by gentle pulse-vortexing on PCR rack 5x | |||
:- Quickly spin down | |||
:- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) | |||
* Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 21ul EB buffer | |||
==== <u>AMPure bead purification</u> ==== | |||
:- Add 60ul AMPure bead. Mix by pipetting 10x | |||
:- Wait for 15min | |||
:- Transfer to sit on magnet for 5min | |||
:- Wash twice with 200ul freshly prepared 80% EtOH | |||
::- After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x | |||
:- Remove all 80% EtOH | |||
:- Dry the bead for 15min | |||
:- Resuspend with 21 EB Buffer | |||
:- Wait for 2min. | |||
:- Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes <u>(discard the beads)</u> | |||
:- Continue to bisulfite conversion | |||
=== Bisulfite conversion === | |||
* Performed bisulfite conversion using the same procedure following manufacturer's instruction [http://www.zymoresearch.com/downloads/dl/file/id/490/d5030i.pdf| '''EZ DNA Methylation-Lightning™ Kit'''] and elute with 34ul Elution buffer. | |||
:- Add 130ul complete Lightning Converson Reagent to 30ul bead-purified adaptor-ligated DNA --> total volume 150ul | |||
:- Mix by pipetting 10X with multi-channel pipette | |||
:- Spin down the tube | |||
:- Incubate following below program (Magprep, Heated Lid) | |||
::- 98°C for 10 minutes (DNA denaturation) | |||
::- 54°C for 1 hours (Bisulfite conversion) | |||
::- 4°C storage for up to 20 hours or continue to desulfonation | |||
:- Add 600ul Binding Buffer to each column | |||
:- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column | |||
:- Mix by pipetting 5x | |||
:- Spin down column at 14,000 rpm for 30s | |||
:- <u>Discard spnt</u> | |||
:- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) | |||
:- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> | |||
:- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube) | |||
:- Wait for 18min | |||
:- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> | |||
:- Add 200ul Wash Buffer | |||
:- Spin down column at 14,000 rpm for 30s | |||
:- <u>Discard spnt</u> | |||
:- Add 200ul Wash Buffer | |||
:- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u> | |||
:- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transfering to new tube | |||
:- Add 36ul of Elution Buffer (warm Elution Buffer at 60C for 10min) | |||
:- Wait for 2min | |||
:- Spin down column at 12,000 rpm for 1min | |||
:- Continue to amplification | |||
== Amplification == | |||
=== TEST PCR === | |||
:- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA |
Revision as of 17:21, 12 December 2014
RRBS library prep, mouse Mouse hippocampus gDNA (Zhou's lab)
- 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab
- I re-quantified concentration by Qubit dsDNA HS assay using 1ul for quantification
Qubit dsDNA HS quantification result
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Conc. (ng/ul) |
HC-C1 | 459 | ng/mL | 1 | 200 | 91.80 |
HC-C2 | 382 | ng/mL | 1 | 200 | 76.40 |
HC-C3 | 510 | ng/mL | 1 | 200 | 100.00 |
HC-C4 | 510 | ng/mL | 1 | 200 | 100.00 |
HC-H1 | 487 | ng/mL | 1 | 200 | 97.40 |
HC-H2 | 495 | ng/mL | 1 | 200 | 99.10 |
HC-H3 | 395 | ng/mL | 1 | 200 | 79.00 |
HC-H4 | 493 | ng/mL | 1 | 200 | 98.60 |
HC-H5 | 428 | ng/mL | 1 | 200 | 85.60 |
1) MspI digestion
- I will use 500ng for RRBS library prep and spike in with 1ng of unmethylated lambda DNA for monitoring bisulfite conversion rate
- Conditions: Digest 500ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 20min
MspI digestion set up
Sample ID | Conc. (ng/ul) | Volume for 500ng (ul) | Sample code | H2O | MspI Mix | Total (ul) |
HC-C1 | 91.80 | 5.45 | C1 | 4.55 | 20.00 | 30.00 |
HC-C2 | 76.40 | 6.54 | C2 | 3.46 | 20.00 | 30.00 |
HC-C3 | 100.00 | 5.00 | C3 | 5.00 | 20.00 | 30.00 |
HC-C4 | 100.00 | 5.00 | C4 | 5.00 | 20.00 | 30.00 |
HC-H1 | 97.40 | 5.13 | H1 | 4.87 | 20.00 | 30.00 |
HC-H2 | 99.10 | 5.05 | H2 | 4.95 | 20.00 | 30.00 |
HC-H3 | 79.00 | 6.33 | H3 | 3.67 | 20.00 | 30.00 |
HC-H4 | 98.60 | 5.07 | H4 | 4.93 | 20.00 | 30.00 |
HC-H5 | 85.60 | 5.84 | H5 | 4.16 | 20.00 | 30.00 |
NTC | 0.00 | 0.00 | NTC | 10.00 | 20.00 | 30.00 |
Msp I reaction Mix
Components | 1 rxn | 10.5 rxn mix |
MspI (10U/ul) | 2.00 | 21.00 |
10X Tango Buffer | 3.00 | 31.50 |
1ng/ul unmeth-lambda DNA | 1.00 | 10.50 |
H2O | 14.00 | 147.00 |
Total | 20.00 |
- - Add H2O to each tube
- - Add 500ng of DNA to each tube
- - Add 20ul of MspI reaction Mix to each tube and pipette few times
- - Spin down the tubes
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tubes
- - 37C, 3h --> 65C, 20min --> hold at 4C (set heat lid)
2) End-repair/dA-tailing
- After MspI, I took 2ul of MspI-digested DNA for gel verification (equal to DNA ~33.33ng). Total volume left is 28ul
- Mix 13ul of 5U/ul Klenow exo- (Thermoscientific) with 13ul of 20:2:2mM dAdCdG
- Add 2ul of Klenow exo-/dAdCdG mix to each tube. Total volume is 30ul
- - Spin down the tubes
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tubes
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- - Perform AMPure bead purification with 2x volume AMPure beads
AMPure bead purification
- - Add 60ul AMPure bead. Mix by pipetting 10x
- - Wait for 15min
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 80% EtOH
- - Dry the bead for 15min
- - Resuspend with 20 EB Buffer (keep the beads in the tubes!)
Methylated adaptor ligation
Components | 1x rxn | 11rxn Mix |
dA-tailed DNA | 20.00 | 0.00 |
10x Ligation Buffer | 3.00 | 33.00 |
T4 DNA Ligase (6000U/ul) | 1.00 | 11.00 |
TruSeq Adaptor (15uM) | 1.50 | 0.00 |
H2O | 4.50 | 49.50 |
Total | 30.00 |
- - Add 1.5ul of 15uM (non-diluted adaptor) TruSeq adaptor to each tube
Sample Truseq Index HC-C1 1 HC-C2 2 HC-C3 3 HC-C4 4 HC-H1 5 HC-H2 6 HC-H3 7 HC-H4 8 HC-H5 9 NTC 10
- Final conc. of adaptor in 30ul lgation reaction is 750nM
- - Add 8.5ul of ligation reaction mix with adaptors
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 24h (no heat lid)
- - Mix by gentle pulse-vortexing on PCR rack 5x
- - Quickly spin down
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 21ul EB buffer
AMPure bead purification
- - Add 60ul AMPure bead. Mix by pipetting 10x
- - Wait for 15min
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 80% EtOH
- - Dry the bead for 15min
- - Resuspend with 21 EB Buffer
- - Wait for 2min.
- - Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- - Continue to bisulfite conversion
Bisulfite conversion
- Performed bisulfite conversion using the same procedure following manufacturer's instruction EZ DNA Methylation-Lightning™ Kit and elute with 34ul Elution buffer.
- - Add 130ul complete Lightning Converson Reagent to 30ul bead-purified adaptor-ligated DNA --> total volume 150ul
- - Mix by pipetting 10X with multi-channel pipette
- - Spin down the tube
- - Incubate following below program (Magprep, Heated Lid)
- - 98°C for 10 minutes (DNA denaturation)
- - 54°C for 1 hours (Bisulfite conversion)
- - 4°C storage for up to 20 hours or continue to desulfonation
- - Add 600ul Binding Buffer to each column
- - Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- - Mix by pipetting 5x
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- - Wait for 18min
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 3min (do not discard spnt)
- - Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transfering to new tube
- - Add 36ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- - Wait for 2min
- - Spin down column at 12,000 rpm for 1min
- - Continue to amplification
Amplification
TEST PCR
- - Prepare PCR in total 20ul reaction with 4 ul BIS-DNA