Noi/NOTES/2013-5-30: Difference between revisions

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** 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH
** 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH
** Incubate at -80C O/N -->
** Incubate at -80C O/N -->
''' Lambda Exonuclease digestion '''<br>
* Resuspend each tube with 100ul H2O, purified with 6X QIAGEN column, eluted 53ul each tube (total volume 318ul, used some for Nanodrop measurement)
* Result
** Conc. dsDNA = 124.5ng/ul, total yield = 315ul * 124.5ng/ul = 39.2ug
''' Removal of amplification adaptors ''' <br>
** Note. The yield of dsDNA amplicons was a little low compare to the previous batch using the same number of PCR reaction, but not too much. This could be due to the number of cycles I used for probe production. In the future, I will try to fix the number of amplification cycle to make it more consistent in each batch.
* Results
** Alternative way for EtOH precipitation, I might precipitate PCR product in 1.5mL tube by pooling 300-400ul of PCR product in each 1.5mL tube  instead of 15mL tube since precipitation in 1.5mL tube is much more efficient than 15mL tube due to the limitation of centrifugation speed. Danial showed me his result by doing this way. He can get double amount of yield in 1.5mL tube compared to 15mL tube. I may need to handle more 1.5mL tube ~12 tubes for half 96-well plate, but I can expect the yield equal to the yield from 96-well reaction by precipitating in 15mL tube. Also, I can save enzyme mix for probe production.
=== Lambda Exonuclease digestion ===
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
| align="center" style="background:#f0f0f0;"|'''2.5x rxn mix'''
|-
| ds amplicons (124.5ng/ul)||125.00||312.50
|-
| 10X Lambda Exo buffer||15.00||37.50
|-
| Lambda Exo (5U/ul)||10.00||25.00
|-
| Total||150.00||375.00
|}
* Aliquot 2X of 150ul and 1X of 75ul,  37C for 1hr
* Purified with 4X QIAGEN column
* Eluted with 40ul EB buffer each (total volume 160ul)
* ssDNA Nanodrop conc. = 73.2ng/ul, yield = 160ul * 73.2ng/ul = 11.7ug
=== Removal of amplification adaptors ===
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1x rxn'''
| align="center" style="background:#f0f0f0;"|'''2x rxn mix'''
|-
| ss amplicons||80.00||160.00
|-
| USER||5.00||10.00
|-
| Total||85.00||170.00
|}
* Aliquot 85ul 2X, 37C for 2hr
* Add 15.5ul of DpnII buffer and RE-DpnII oligos mix (mix 10.5ul of 10X DpnII buffer and 5ul of 100uM RE-DpnII oligo guide) toeach tube
* 94C for 3min -> 37 C 3min
* Add 5ul of DpnII, mix by pipetting 10 times on thermocycler
* 37 C for 2 hr
* Add 95 ul of USER enzyme mix (5ul USER, 10ul of 10X DpnII buffer, 80ul H2O
* 37C for 2hr
* Heat inactivate enzyme at 75C for 20min (optional)
''' PAGE verification before column purification '''<br>
''' PAGE verification before column purification '''<br>
  [[File:ZhangLab_2 2013-06-01 16hr 06min_Blueprint_PAGEverification.jpg| 200px]]
  [[File:ZhangLab_2 2013-06-01 16hr 06min_Blueprint_PAGEverification.jpg| 200px]]
 
Image descriptions:
1: ds amplicons
2: ss amplicons after lambda exonuclease digestion
3: USER digested ssDNA
4: USER -> DpNII digested ssDNA
5: USER -> DpnII -> USER digested ssDNA
* Note: For lane 1-3, I took different volume of reaction to get the same amount of DNA in each lane to compare the efficiency of digestion by each enzyme
* Purified with 4X QIAGEN column.
* Eluted with 52ul EB buffer each (total volume = 208ul)
* ssDNA Nanodrop = 31.7ng/ul, yield = 206ul * 31.7ng/ul = 6.53ug
''' PAGE size-selection '''<br>
''' PAGE size-selection '''<br>
  [[File:ZhangLab_2 2013-06-03 12hr 13min_PAGE-SS_Blueprint.jpg| 400px]] [[File:ZhangLab_2 2013-06-03 12hr 09min_PAGE-SS_Blueprint.jpg| 200px]]
* Did PAGE size-selection in 3X 2D-well TBU gel, by loading ~2.2ug DNA/gel
  [[File:ZhangLab_2 2013-06-03 12hr 13min_PAGE-SS_Blueprint.jpg| 400px]] [[File:ZhangLab_2 2013-06-03 12hr 09min_PAGE-SS_Blueprint.jpg| 186px]]
* Purified in 6X 1.5 mL lo-bind tube and resuspend each tube with 11ul H2O (total volume ~66ul)
Image descriptions:
''' PAGE quantification of purified BSPP Blueprint '''<br>
''' PAGE quantification of purified BSPP Blueprint '''<br>
  [[File:ZhangLab_2 2013-06-04 13hr 22min_BSPP_BP_PQ2.jpg| 400px]]
- I diluted 2ul of BSPP Bluprint probe with 38ul H2O then added 40ul of 2X tBU loading dye.
- I then loaded different volume of dilute probe in 6% TBU gel (3, 4, 5, 6, 7, 8,& 10ul)
- For LM ladder, I mixed 2ul of 1ug/ul ladder in total 20ul, and loaded 4, 6, & 8ul
  [[File:ZhangLab_2 2013-06-04 13hr 22min_BSPP_BP_PQ2.jpg| 350px]]
Image descriptions:
- The number is the loading volume of diluted probe (2ul in total 80ul).
3 = 0.075 ul
4 = 0.10 ul
5 = 0.125 ul
6 = 0.150 ul
7 = 0.175 ul
8 = 0.20 ul
10 = 0.25 ul
- The concentration of the probe is very high, so the volume > 6ul (actual volume 0.15) gave a saturate signal
* Total volume of probe 65ul, conc. by PAGE quantification: 48.2ng/ul, total amount = 65ul * 48.2ng/ul = 3.1ug (from total 48 PCR reaction)
* Total volume of probe 65ul, conc. by PAGE quantification: 48.2ng/ul, total amount = 65ul * 48.2ng/ul = 3.1ug (from total 48 PCR reaction)
 
* For accuracy of probe concentration quantification, I currently use Qubit ssDNA assay.
''' 2013 - 06 - 03 '''
''' 2013 - 06 - 03 '''
* Dinh received H1 gDNA passage #36 (conc. 149ng/ul, total amount ~8ug --> total volume ~ 52ul) from Belmonte's lab.  
* Dinh received H1 gDNA passage #36 (conc. 149ng/ul, total amount ~8ug --> total volume ~ 52ul) from Belmonte's lab.  
Line 133: Line 200:
* In the future, I will use Qubit to measure gDNA concentration for BSPP experiment to reduce the inaccurate of DNA concentration due to the impurity of DNA samples.
* In the future, I will use Qubit to measure gDNA concentration for BSPP experiment to reduce the inaccurate of DNA concentration due to the impurity of DNA samples.
* It seemed like I will not have much gDNA to test for Blueprint project
* It seemed like I will not have much gDNA to test for Blueprint project
* Dr. Zhang suggested to not restrict to H1 DNA sample, but I can use any gDNA sample from HAPMAP plate since the 450K Illumina methylation array data are available and it should cover the assigned CpG sites.
----
Continued on: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-6-6]]

Latest revision as of 15:43, 6 May 2014

DNA methylation assay for Blueprint project[edit]

2013-05-30

  • Received oligos from CustomArray
  • Oligo information
    • Design: BSPP_Design-5-17-2013 --> I would name BSPP_Blueprint for future experiment
    • Length 150 mer (MW = 150 * 330Da/bp = 49,500 Da or g/mole
    • Cocn. 40.16ng/ul = 811.3 nM (40.16ng/ul/ 49,500 g/mole)
    • Volume 80ul in TE buffer. Total amount 3.21ug.
  • Probe prep plan
    • Last time of Matt's experiment on Expansion PCR of oligos from CustomArray, we need to use template ~100x more than oligos synthesized from Agilent. This time, I will test expansion PCR using different concentration of oligo template (concentration in expansion PCR 20nM and 100nM) and use DMR330k oligos (Exp1) from Agilent as PC.
    • Aliquot 40ul oligo from original tube and dilute to 500nM (mix 40ul of 811.3nM oligo with 24.90ul H2O)

Expansion PCR (TEST)[edit]

Components Volume (ul) Final conc. Volume for 3.5 rxn mix
500nM seed oligoes 2.00 or 5.00 20nM or 50nM 0.00
AP1V6 (100uM) 0.20 400nM 0.70
AP2V6 (100uM) 0.20 400nM 0.70
2x KAPA SYBG fast MM 25.00 1x 87.50
H2O 19.60 68.60
Total 45.00 157.50
  • Aliquot 45ul, add oligo template, and adjust total volume to 50ul with H2O
  • Add 2.5 ul of DMR330k Exp1, final conc. in expansion PCR is 1nM
Program

95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold
File:20130530 BSPPBlueprint TESTExpensionPCR.png
  • from qPCR curve, seemed like 20nM and 50nM didn't amplify very well comparing to PC as expected (similar to Matt's result using the oligos from the same source), so after PAGE verification, I will do expansion PCR with 100nM template.

PAGE analysis to verify amplified amplicons in 6% TBU gel

  • Loaded 2.5ul of PCR products in TBU gel
  • I loaded amplified amplicons in TBU gel becuase I dodn't want to waste the TBU gel used for single strand seed oligos verification
File:ZhangLab 2 2013-05-30 15hr 54min TESTExpansionPCR.jpg

Lane1: 10bp DNA ladder
Lane2: O = 3ul of 500nM oligo before expansion PCR
Lane3: PC (BSPP DMR330k Exp1)
Lane4: 20nM BSPP _Blueprint after expansion PCR
Lane5: 50nM BSPP _Blueprint after expansion PCR
  • From the gel image above, the expected band of ~150 bp was amplified.
  • The seed oligos from original tube before expansion PCR show a smear as observed in Matt' oligos.
  • There was the extra band in PC of DMR330k Exp1 >1kb. I have no idea where it come from. I opened this tube only twice, but every time I opened in the hood. I observed a faint band in the reaction containing BSPP Blueprint oligo with slightly larger size.
  • I continued to do expansion PCR by using oligo in the reaction 100nM, 2 tubes of 100ul reaction

Expansion PCR[edit]

Components Volume (ul) Final conc.
500nM seed oligoes 40.00 100nM
AP1V6 (100uM) 0.80 400nM
AP2V6 (100uM) 0.80 400nM
2x KAPA SYBG fast MM 100.00 1x
H2O 58.40
Total 200.00
  • Split into 2 tubes (100ul each)
Program

95C 30sec -> (95C 30sec -> 54C 45sec-> 72C 45sec) X 15-> 72C 3min -> 15C hold

** I stopped at 12 cycles
File:20130530 BSPPBlueprint ExpensionPCR.png
  • Purified with Qiaquick column (2 columns) and elute with EB buffer volume 40ul each --> total volume = 80ul
  • Measure DNA conc. with Nanodrop: 17.4ng/ul or 175.7nM
  • M.W. of dsDNA 150bp = 150bp * 660Da/bp = 99,000 g/mole
  • Dilute 1st round amplicon to 10nM volume 400 ul (mix 22.77ul of 175.7nM 1st round amplicons with 377.23 ul H2O) for using as the template for future amplification
  • Performed production PCR

Production PCR[edit]

  • Since I will do trial experiment before do capture on the real samples, I would prepare more probes to make sure that it is enough for entire project.
Components 1 rxn 50x rxn mix
1st round amplicon (10nM) 0.20 10.00
AP1V61U (100uM) 0.40 20.00
AP2V6 (100uM) 0.40 20.00
2x KAPA SYBG fast MM 50.00 2,500.00
H2O 49.00 2,450.00
Total 100.00 5,000.00
  • Aliquot 100ul to each tube in 6X 8-strip tubes
Program

95C 30sec -> (95C 10sec -> 55C 20sec-> 72C 30sec) X 15-> 72C 3min -> 15C hold
File:20130530 ProductionPCR BSPPBlueprint.png
  • EtOH precipitation of the 48 rxn PCR in 6x 15mL tube
    • 800ul amplified amplicons + 80ul 3M NaOAc pH5.5 + 2.6ul Glycoblue and 2mL 100% EtOH
    • Incubate at -80C O/N -->
  • Resuspend each tube with 100ul H2O, purified with 6X QIAGEN column, eluted 53ul each tube (total volume 318ul, used some for Nanodrop measurement)
    • Conc. dsDNA = 124.5ng/ul, total yield = 315ul * 124.5ng/ul = 39.2ug
    • Note. The yield of dsDNA amplicons was a little low compare to the previous batch using the same number of PCR reaction, but not too much. This could be due to the number of cycles I used for probe production. In the future, I will try to fix the number of amplification cycle to make it more consistent in each batch.
    • Alternative way for EtOH precipitation, I might precipitate PCR product in 1.5mL tube by pooling 300-400ul of PCR product in each 1.5mL tube instead of 15mL tube since precipitation in 1.5mL tube is much more efficient than 15mL tube due to the limitation of centrifugation speed. Danial showed me his result by doing this way. He can get double amount of yield in 1.5mL tube compared to 15mL tube. I may need to handle more 1.5mL tube ~12 tubes for half 96-well plate, but I can expect the yield equal to the yield from 96-well reaction by precipitating in 15mL tube. Also, I can save enzyme mix for probe production.

Lambda Exonuclease digestion[edit]

Components 1x rxn 2.5x rxn mix
ds amplicons (124.5ng/ul) 125.00 312.50
10X Lambda Exo buffer 15.00 37.50
Lambda Exo (5U/ul) 10.00 25.00
Total 150.00 375.00
  • Aliquot 2X of 150ul and 1X of 75ul, 37C for 1hr
  • Purified with 4X QIAGEN column
  • Eluted with 40ul EB buffer each (total volume 160ul)
  • ssDNA Nanodrop conc. = 73.2ng/ul, yield = 160ul * 73.2ng/ul = 11.7ug

Removal of amplification adaptors[edit]

Components 1x rxn 2x rxn mix
ss amplicons 80.00 160.00
USER 5.00 10.00
Total 85.00 170.00
  • Aliquot 85ul 2X, 37C for 2hr
  • Add 15.5ul of DpnII buffer and RE-DpnII oligos mix (mix 10.5ul of 10X DpnII buffer and 5ul of 100uM RE-DpnII oligo guide) toeach tube
  • 94C for 3min -> 37 C 3min
  • Add 5ul of DpnII, mix by pipetting 10 times on thermocycler
  • 37 C for 2 hr
  • Add 95 ul of USER enzyme mix (5ul USER, 10ul of 10X DpnII buffer, 80ul H2O
  • 37C for 2hr
  • Heat inactivate enzyme at 75C for 20min (optional)

PAGE verification before column purification

File:ZhangLab 2 2013-06-01 16hr 06min Blueprint PAGEverification.jpg

Image descriptions:
1: ds amplicons
2: ss amplicons after lambda exonuclease digestion
3: USER digested ssDNA
4: USER -> DpNII digested ssDNA
5: USER -> DpnII -> USER digested ssDNA
* Note: For lane 1-3, I took different volume of reaction to get the same amount of DNA in each lane to compare the efficiency of digestion by each enzyme
  • Purified with 4X QIAGEN column.
  • Eluted with 52ul EB buffer each (total volume = 208ul)
  • ssDNA Nanodrop = 31.7ng/ul, yield = 206ul * 31.7ng/ul = 6.53ug

PAGE size-selection

  • Did PAGE size-selection in 3X 2D-well TBU gel, by loading ~2.2ug DNA/gel
File:ZhangLab 2 2013-06-03 12hr 13min PAGE-SS Blueprint.jpg File:ZhangLab 2 2013-06-03 12hr 09min PAGE-SS Blueprint.jpg
  • Purified in 6X 1.5 mL lo-bind tube and resuspend each tube with 11ul H2O (total volume ~66ul)
Image descriptions:

PAGE quantification of purified BSPP Blueprint

- I diluted 2ul of BSPP Bluprint probe with 38ul H2O then added 40ul of 2X tBU loading dye. 
- I then loaded different volume of dilute probe in 6% TBU gel (3, 4, 5, 6, 7, 8,& 10ul)
- For LM ladder, I mixed 2ul of 1ug/ul ladder in total 20ul, and loaded 4, 6, & 8ul

File:ZhangLab 2 2013-06-04 13hr 22min BSPP BP PQ2.jpg

Image descriptions: 
- The number is the loading volume of diluted probe (2ul in total 80ul). 
3 = 0.075 ul
4 = 0.10 ul
5 = 0.125 ul
6 = 0.150 ul
7 = 0.175 ul 
8 = 0.20 ul
10 = 0.25 ul
- The concentration of the probe is very high, so the volume > 6ul (actual volume 0.15) gave a saturate signal

  • Total volume of probe 65ul, conc. by PAGE quantification: 48.2ng/ul, total amount = 65ul * 48.2ng/ul = 3.1ug (from total 48 PCR reaction)
  • For accuracy of probe concentration quantification, I currently use Qubit ssDNA assay.

2013 - 06 - 03

  • Dinh received H1 gDNA passage #36 (conc. 149ng/ul, total amount ~8ug --> total volume ~ 52ul) from Belmonte's lab.
  • I re-quantify concentration by Nanodrop and Qubit dsDNA HS assay to confirm concentration and to compare the concentration between Nanodrop and Qubit.
    • Nanodrop : 100.36 ng/ul, 260/280 = 2.07, 260/230 = 2.75
    • Qubit dsDNA HS assay: 23 ng/ul (used 1ul of sample for quantification)
  • The concentration of H1 gDNA sample by Nanodrop and Qubit quantification is ~4X different between Nanodrop and Qubit. This might be the reason that I got very low yield after bisulfite conversion even I started with high amount of gDNA (1 - 1.2ug by Nanodrop before bisulfite conversion and measure bisulfite converted gDNA by Qubit ssDNA assay) . Note that in the past, I relied on the concentration by Nanodrop measurement since it's very convenient to measure many samples than Qubit.
  • I don't think it come from the calibration of machine since I used to measure standard DNA used for Qubit quantification (has very high purity) and Nanodrop, I got very close concentration by the two measurement. It should be the impurity in DNA that interfere 260nm absorbance.
  • In the future, I will use Qubit to measure gDNA concentration for BSPP experiment to reduce the inaccurate of DNA concentration due to the impurity of DNA samples.
  • It seemed like I will not have much gDNA to test for Blueprint project
  • Dr. Zhang suggested to not restrict to H1 DNA sample, but I can use any gDNA sample from HAPMAP plate since the 450K Illumina methylation array data are available and it should cover the assigned CpG sites.

Continued on: [[1]]