Matt:LabNotes/2015-1-26: Difference between revisions
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>Mzcai (Created page with "==CA12k_Nov2014 RevComp Probe Production== *Production of Padlock Probes targeting cDNA (V6 and V8 primers) of Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering...") |
>Mzcai mNo edit summary |
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'''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold | '''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold | ||
[[File: | [[File:012614_CA12kNov14_ExpansionPCR_V6V8.JPG]] | ||
*Purified each with 1 Qiagen column and eluted with 50ul H2O | *Purified each with 1 Qiagen column and eluted with 50ul H2O | ||
*Nanodrop: | *Nanodrop: | ||
** | **V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM | ||
** | **V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM | ||
*Diluted to 10nM | *Diluted to 10nM | ||
** | **V6: 48ul x 95nM = '''456ul''' x 10nM | ||
***Add | ***Add 408ul H2O | ||
**V7: 50ul x | **V8: 48ul x 129nM = '''619ul''' x 10nM | ||
***Add | ***Add 571ul H2O | ||
====Gel Check==== | |||
*Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye | |||
*Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control | |||
<!-- | |||
[[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]]--> |
Revision as of 22:27, 26 January 2015
CA12k_Nov2014 RevComp Probe Production
- Production of Padlock Probes targeting cDNA (V6 and V8 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:
- V6 primers - CA12k_Nov2014_Contig_RevComp
- V8 primers - CA12k_Nov2014_Const_RevComp
' | Contig of Exons RevComp(V6) | Constitutive Exons RevComp(V8) |
CA12k_Nov2014 Seed Oligos (900nM) | 15 | 15 |
2X KAPA SYBG MM | 67.5 | 67.5 |
100uM AP1V6U | 0.6 | 0 |
100uM AP2V6 | 0.6 | 0 |
100uM AP1V8U | 0 | 0.6 |
100uM AP2V8 | 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:012614 CA12kNov14 ExpansionPCR V6V8.JPG
- Purified each with 1 Qiagen column and eluted with 50ul H2O
- Nanodrop:
- V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM
- V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM
- Diluted to 10nM
- V6: 48ul x 95nM = 456ul x 10nM
- Add 408ul H2O
- V8: 48ul x 129nM = 619ul x 10nM
- Add 571ul H2O
- V6: 48ul x 95nM = 456ul x 10nM
Gel Check
- Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
- Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control