Editing
Matt:LabNotes/2015-1-5
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_Nov2014 Probe Production== *Production of Padlock Probes targeting cDNA (V4 and V7 primers) of [[Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering]] ===Expansion PCR=== *[[Matt:LabNotes/2014-12-18]] conclusion: 21 cycles of PCR starting with 100nM seed oligos *Make 2 tubes: **V4 primers - CA12k_Nov2014_Contig **V7 primers - CA12k_Nov2014_Const {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Contig of Exons (V4)''' | align="center" style="background:#f0f0f0;"|'''Constitutive Exons (V7)''' |- | CA12k_Nov2014 Seed Oligos (900nM)||15||15 |- | 2X KAPA SYBG MM||67.5||67.5 |- | 100uM AP1V4U||0.6||0 |- | 100uM AP2V4||0.6||0 |- | 100uM AP1V7U||0||0.6 |- | 100uM AP2V7||0||0.6 |- | H2O||51.3||51.3 |- | Total||135||135 |} '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold [[File:010514_CA12kNov14_ExpansionPCR_V4V7.JPG]] *Purified each with 1 Qiagen column and eluted with 50ul H2O *Nanodrop: **V4: 10.8 ng/ul -> 10.8 ng/ul / (188bp*660Da/bp) = 87nM **V7: 17.8 ng/ul -> 17.8 ng/ul / (190bp*660Da/bp) = 142nM *Diluted to 10nM **V4: 50ul x 87nM = '''435ul''' x 10nM ***Add 385ul H2O **V7: 50ul x 142nM = '''710ul''' x 10nM ***Add 660ul H2O ====Gel Check==== *Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye *Use 10nM Agi26k0gap expansion PCR'd probes as positive control *Although band is '''very''' faint it can be seen at slightly under 200bp [[File:2015-01-06_1stRndAmplicon_V4V7_Gel.jpg]] ===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_Nov14_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 |} *V7 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_Nov14_V7 (10nM)||0.2||10 |- | 2X KAPA SYBG MM||50||2500 |- | AP1V7U (100uM)||0.4||20 |- | AP2V7 (100uM)||0.4||20 |- | H2O||49||2450 |- | Total||100||5000 |} '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold *Stopped after 15 cycles [[File:010514_CA12kNov14_ProductionPCR_V4V7.JPG | 800px]] ===EtOH Precipitation=== *6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7 **800ul PCR product **2000ul 100% EtOH **2.7ul GlycoBlue **80ul 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 12 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 100ul H2O ===Qia Column Purification=== *Purified 12 tubes of 100ul in 12 columns following Qiagen protocol *Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another *Measured concentration with Nanodrop: ~290ul of V4 probes: 127.8 ng/ul => ~37 ug ~290ul of V7 probes: 113.9 ng/ul => ~33 ug ===Lambda Exonuclease Digestion=== *Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Amplicon||72.5 |- | 10X Lambda Exo Buffer||10 |- | Lambda Exonuclease||10 |- | H2O||7.5 |- | Total||100 |} *Incubated at 37C for 1hr *Purified with 4 Zymo ssDNA/RNA columns *Eluted with 40ul each *Recombined and measured ssDNA with Nanodrop: **66.5ng/ul x 160ul = 10.6ug (57.3% yield) **56.9ng/ul x 160ul = 9.1ug (55.2% yield) ===Remove Amplification Adapters=== ====USER==== *Split each set into 2 PCR tubes and add 5ul USER {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | SS-amplicon||80 |- | USER||5 |- | Total||85 |} *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||10 |- | 100uM RE-DpnII guide oligo||5 |} RE-DpnII guide oligo: V4: RE DpnII V4 V7: RE DpnII V7 *Incubated at 94C for 2 min, then 37C for 3 min *Added 5ul DpnII *Incubated at 37C for 4 hr ===TBU Gel: Digestion Check=== *Combine into 1 tube for each set *2ul Digested Sample + 2ul 2X Urea Loading Dye *5ul Undigested sample (10nM 1st round amplicon) + 5ul 2X Urea Loading Dye *0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye [[File:2015-01-07_V4V7_Digest_GelCheck.jpg]] *Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters *To get a higher yield I continued digestion overnight ===Continue Digestion=== *Add 100ul of the following to each tube {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X DpnII Buffer||10 |- | USER||5 |- | H2O||85 |- | Total||100 |} *Incubated at 37C for overnight (15 hrs) ===Zymo Column Purification=== *4 columns for each set *Eluted 30ul each column (120ul total) *Nanodrop **V4: 45.0ng/ul x 120ul = 5.4ug **V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice) ===PAGE Size Selection=== *Run 2 gels for V4 and 1 gel for V7 **220V for 30min {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | V4 Probes||120 |- | TBE-Urea Buffer 2X||120 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' |- | V7 Probes||120 |- | TBE-Urea Buffer 2X||120 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Low Mass Ladder||3 |- | TBE-Urea Buffer 2X||15 |- | H2O||12 |- | Total||30 |} *V4 Gel 1 [[File:2015-01-08_SizeSelect_V4_Gel1.jpg]] *V4 Gel 2 [[File:2015-01-08_SizeSelect_V4_Gel2.jpg]] *V7 Gel 1 **Had trouble seeing blade and may have cut some extra above where I should have. However a mitigating factor is that V7 was digested much more completely than V4 based on intensity of band before cutting [[File:2015-01-08_SizeSelect_V7_Gel1.jpg]] ===EtOH Precipitation=== *Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT. *Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip. *Added 900 ul of 1X TE buffer *Vortexed for 60 min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transfered the clear spnt. to 6 Nanosep column and centrifuged at 15,000 rpm for 3 min *Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transfered spnt to fresh 1.5 mL tube (~400ul per tube) *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 *Vortexed and placed the 6 tubes at -80C for overnight *Spun 6 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul 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 recombine (40ul for V4 and 20ul for V7) ===TBU Gel Quantification=== *Dilute 2ul to 10ul with H2O *Add 10ul 2X TBE-Urea Buffer *Load wells with 2, 4, 6, and 8 ul *Load Low Mass DNA ladders in 0.5, 1, and 2 ul *Ran at 200 V for 35min {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0.5 ladder''' | align="center" style="background:#f0f0f0;"|'''1 ladder''' | align="center" style="background:#f0f0f0;"|''' 2 ul ''' | align="center" style="background:#f0f0f0;"|''' 4 ul ''' | align="center" style="background:#f0f0f0;"|''' 6 ul ''' | align="center" style="background:#f0f0f0;"|''' 8 ul ''' | align="center" style="background:#f0f0f0;"|'''2 ladder''' |- | DNA volume||0.5||1||0.1||0.2||0.3||0.4||2 |- | H2O||1.5||1||0.9||1.8||2.7||3.6||0 |- | 2X Buffer||2||2||1||2||3||4||2 |- | Total||4||4||2||4||6||8||4 |- | |} [[File:2015-01-09_V4_GelQuant.jpg|650px]] [[File:2015-01-09_V7_GelQuant.jpg|650px]] *Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear *V4: 10.7 ng/ul => 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul) *V7: 6.2 ng/ul => 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul) [[Media:2015-01-09_V4V7_GelQuant.xlsx | How I calculated concentration]]
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