Editing
Noi/NOTES/2014-5-31
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
= DNA extraction of 10 serum samples from Cancer Center = * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] * I extract 10 serum samples from cancer patients received on 2014-05-27 [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:Samples_from_Moores_Cancer_Center]] * There are two tubes of serum. Dr. Zhang suggested me to extract DNA on one tube first to see the result from the first tube and save the second tube in case there is something wrong with the first experiment * I assigned new sample codes to be more convenient for sample handling. * I measured volume of the first tube serum during DNA extraction and adjust volume to 400ul with 1X PBS as ChargeSwitch kit required different volume of reagent for different sample volume. ** Note that the measurement could be +-5ul error since serum is viscous and I needed to adjust the pipette volume after aspiration to measure the volume :{| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID #''' | align="center" style="width:120px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume tube_1''' | align="center" style="width:100px;background:#f0f0f0;"|'''PBS to 400ul''' |- | UCSD-004-04||PC-S_1||300||100 |- | UCSD-004-05||PC-S_2||305||95 |- | UCSD-004-06||PC-S_3||165||235 |- | UCSD-004-07||PC-S_4||250||150 |- | UCSD-004-08||PC-S_5||315||85 |- | UCSD-004-09||PC-S_6||315||85 |- | UCSD-004-10||PC-S_7||300||100 |- | UCSD-004-11||PC-S_8||180||220 |- | UCSD-004-12||PC-S_9||250||150 |- | UCSD-004-13||PC-S_10||250||150 |} * '''PC = pancreatic cancer''' *'''S = serum smaple''' * I followed instruction of ChargeSwith gDNA 1 ml Serum kit as described in [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-11#3._ChargeSwitch.C2.AE_gDNA_1_ml_Serum_kit]] for 400ul sample volume '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> :{| {{table}} | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''10.5x''' |- | Lysis Buffer||280||2,940 |- | Proteinase K||30||315 |} :''- Aliquot 310ul'' :{| {{table}} | align="center" style="width:180px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x''' | align="center" style="width:80px;background:#f0f0f0;"|'''10.5x''' |- | Purification Buffer (N7)||100||1,050 |- | Magnetic beads||12||126 |} :''- Aliquot 112ul'' * Eluted 30ul with Elution Buffer * Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution) ==== Qubit dsDNA HS assay result ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:100px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:100px;background:#f0f0f0;"|'''Sample Conc. (ng/ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield (ng)''' |- | PC-S_1||8.79||ng/mL||2||100||0.879||30||26.37 |- | PC-S_2||16.6||ng/mL||2||100||1.660||30||49.80 |- | PC-S_3||214||ng/mL||2||100||21.400||30||642.00 |- | PC-S_4||8.5||ng/mL||2||100||0.850||30||25.50 |- | PC-S_5||15.8||ng/mL||2||100||1.580||30||47.40 |- | PC-S_6||175||ng/mL||2||100||17.500||30||525.00 |- | PC-S_7||18||ng/mL||2||100||1.800||30||54.00 |- | PC-S_8||9.53||ng/mL||2||100||0.953||30||28.59 |- | PC-S_9||57.2||ng/mL||2||100||5.720||30||171.60 |- | PC-S_10||17.1||ng/mL||2||100||1.710||30||51.30 |} * I may need to repeat sample PC-S_3 andPC-S_6 as the concentration is very high. To save DNA, I will use 1ul for the assay. ** I have repeated measuring conc. of PC-S-3 and PC-S-4 and the conc. are 17.2 and 14.7 ng/ul, respectively. These numbers are close to previous measurement. I will use previous numbers as they were measured in the same time of other samples. ==== Normalize DNA yield to 1mL serum volume ==== {| {{table}} class = wikitable | align="center" style="width:150px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:100px;background:#f0f0f0;"|'''Yield (ng)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Serum volume (ul)''' | align="center" style="width:100px;background:#f0f0f0;"|'''Normalized yield to 1mL (ng)''' |- | PC-S_1||26.37||300||87.90 |- | PC-S_2||49.80||305||163.28 |- | PC-S_3||642.00||165||3,890.91 |- | PC-S_4||25.50||250||102.00 |- | PC-S_5||47.40||315||150.48 |- | PC-S_6||525.00||315||1,666.67 |- | PC-S_7||54.00||300||180.00 |- | PC-S_8||28.59||180||158.83 |- | PC-S_9||171.60||250||686.40 |- | PC-S_10||51.30||250||205.20 |} * Some samples have the yield lower than 50ng. I am afraid that this amount may not be enough for BSPP capture. After bisulfite conversion the yield might be only 10ng or lower. * I need to repeat V1N3 capture to confirm the minimum amount of BIS-DNA required for capture * For current experiment, the minimum is 50ng. This is still higher than the amount of some non-bisulfite converted samples from this batch
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information