Editing
Matt:LabNotes/2016-11-2
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!
=CA12k_Oct2016 Probe Preparation= *Received CA12k_Oct2016_Oligos2Order_FINAL in 80ul TE Buffer **47.35ng/ul (3.79ug total) ***Calculate MW of 195 nt probe = 195nt * 303.7D/nt + 79D = 59,300.5 g/mol **47.35 ng/ul * nmol/59300.5ng = 0.0007985 nmol/ul = 0.8 pmol/ul = 0.8 uM *Make 12.5ul aliquots in PCR strip tubes for future *[[Matt:LabNotes/2016-10-14|CA12k_Oct2016 Probe Design]] **May also see this probe set called TB12k_Oct2016 in my notes {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Probeset | width="51" | Amplification | width="51" | # Probes | width="51" | # Barcodes | width="51" | # Genes |- style="font-size:11pt" valign="bottom" | height="14" | Human Brain | V4 | align="right" | 4,978 | align="right" | 391 | align="right" | 368 |- style="font-size:11pt" valign="bottom" | height="14" | Mouse Brain | V7 | align="right" | 3,602 | align="right" | 342 | align="right" | 319 |- style="font-size:11pt" valign="bottom" | height="14" | Mouse Embryo | V6 | align="right" | 2,469 | align="right" | 251 | align="right" | 220 |- style="font-size:11pt" valign="bottom" | height="14" | Lung Cancer | V8 | align="right" | 105 | align="right" | 105 | align="right" | 85 |} ==Expansion PCR Test V4== *NTC {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Seed oligo||0 |- | F/R V4 Primer Mix (10uM)||2 |- | 2X Kapa MM||12.5 |- | H2O||10.5 |- | Total||25 |} *Pos Control {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | TB12k_Apr2016 Seed oligo (100X Dilution)||2.5 |- | F/R V4 Primer Mix (10uM)||2 |- | 2X Kapa MM||12.5 |- | H2O||8 |- | Total||25 |} *50nM CA12k_Oct2016 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Seed oligo (800nM)||1.5 |- | F/R V4 Primer Mix (10uM)||2 |- | 2X Kapa MM||12.5 |- | H2O||9 |- | Total||25 |} *100nM CA12k_Oct2016 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Seed oligo (800nM)||3.125 |- | F/R V4 Primer Mix (10uM)||2 |- | 2X Kapa MM||12.5 |- | H2O||7.375 |- | Total||25 |} '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold [[File:20161102_CA12kOct2016V4_ExpansionPCRtest.JPG|450px]] ==Expansion PCR V4== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Seed oligo (800nM)||12.5 |- | F/R V4 Primer Mix (10uM)||8 |- | 2X Kapa MM||50 |- | H2O||29.5 |- | Total||100 |} *50ul per PCR tube '''Program''' 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 23 -> 72C 2min -> 15C hold [[File:20161102_CA12kOct2016V4_ExpansionPCR.JPG|450px]] *Purified with 2 Qiagen PCR columns (50ul PCR product each) **Elute with 50ul H2O *Quantify with Qubit dsDNA: **2.6ng/ul / (195bp*607.4Da/bp+157.9Da) = 21.9 nmol/L *Dilute to 10nM **21.9nM x 99ul = 10nM x 216.8ul **Add 117.8ul H2O ==Production PCR== *V4 master mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (1X)''' | align="center" style="background:#f0f0f0;"|'''Volume (50X)''' |- | First round amplicon CA12k_Oct2016_V4 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V4U (100uM)||0.4||20 |- | AP2V4 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} *Did one plate - 96 wells 50ul each '''Program''' 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold [[File:20161103_CA12kOct2016V4_ProductionPCR.JPG|450px]] *Stopped after 14 cycles **Based on touchscreen image curve probably peaked at cycle 13 and started to go down just a little at cycle 14 *Around cycle 12 the PCR curve started to look tilted like in the image here **It started with about half the wells with no obvious pattern which wells **Right at cycle 14 all the remaining normal curves also became tilted ===EtOH Precipitation=== *8 5-ml tubes each with **600ul PCR product **3000ul 100% EtOH **4ul GlycoBlue **120ul 3M NaOAc pH 5.2-5.5 *Vortexed and put in -80C for overnight *Centrifuged at 3000rpm at 4C for 30 min *Discarded supernatant and added 800ul of cold 80% EtOH *Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes *Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C *Discarded supernatant and air-dried for 5 min in hood *Resuspended DNA with 50ul H2O (usually 100ul but because only did 5ml instead of 10ml production PCR this time) ===Qia Column Purification=== *4 columns elute 50ul each *Nanodrop: 208.8 ng/uL x 200uL = 41.76ug ===Lambda Exo=== *Divide into 4 pcr tubes of 100ul (ideally should be <10ug each) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||50 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||30 |- | Total||100 |} *Incubated at 37C for 1hr *Purified with 4 Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **96.8ng/ul x 160ul = 15.49ug (74% yield) ===Remove Amplification Adapters=== *Save 4ul for gel check of size ====USER==== *Split into 3 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||52 |- | USER||5 |- | 10X DpnII Buffer||8 |- | H2O||15 |- | Total||80 |} *Incubate at 37C for 2.5 hours ====DpnII==== *Added 15ul of the following {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||2 |- | 100uM RE-DpnII_V4 guide oligo||5 |- | H2O||8 |- |} *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for overnight (~15hrs) ===Zymo Column Purification=== *Eluted 20ul each column (60ul total) *Nanodrop **87.3 ng/ul x 60ul = 5.2ug (34% yield lower than normal) ===Gel Check=== *Pre-run 12 well TBU gel for 20min at 250V *Rinse wells #Ladder #*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X TBU Loading Dye #User/DpnII-Digested #*2ul Sample + 2ul 2X TBU Loading Dye #Lambda-Digested #*2ul Sample + 2ul 2X TBU Loading Dye *Pre-heat samples to 75C for 10min and then put on ice *Load samples in middle lanes of gel *Run for 33min at 210V [[File:2016-11-08_CA12kOct2016_V4_GelCheck.jpg|250px]] ===PAGE Size Selection=== *Run 2 gels *200V for 40min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | V4 Probes||60 |- | TBE-Urea Buffer 2X||60 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | Low Mass Ladder||2 |- | TBE-Urea Buffer 2X||10 |- | H2O||8 |- | Total||20 |} [[File:|350px]]<br> [[File:|350px]]<br> ===EtOH Precipitation=== *Put cut out gel in 4 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT. *Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. *Added 450 ul of 1X TE buffer to each *Vortexed for 60min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min *Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transferred spnt to fresh 1.5 mL tube *Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2 *Vortexed and placed the 4 tubes at -80C for 30min *Spun 4 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 10ul and combine ===Qubit ssDNA=== 990pg/ul x 40ul = 39.6ng 990pg/ul / (157 x 303.7Da/nt + 79Da) = 20.7nM
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