Daniel:Notebook/ComboLock/2016-6-29: Difference between revisions
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===qPCR Results=== | ===qPCR Results=== | ||
<gallery perrow=2 heights=200px widths=200px> | |||
File:VIM-Ab-LPTest2_Results_20160629.png|qPCR Curves | |||
File:2016-06-29-CombolockAb-LP-Test2.jpg|Gel image | |||
</gallery> | |||
=C Probe SOD1/VIM Test= | =C Probe SOD1/VIM Test= |
Revision as of 22:52, 2 July 2016
Antibody Stage 2 Test 2 (Started yesterday)
Protcol
- Latch and Padlock Hybridization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
- qPCR
- Set up reaction according to table
- Master Mix
- 165 uL KAPA SyberFast MM
- 115.5 uL nfH2O
- 3.3 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
- VIM-Ab-LPTest2 Results 20160629.png
qPCR Curves
- 2016-06-29-CombolockAb-LP-Test2.jpg
Gel image
- VIM PA.png
VIM PA probe
- VIM PB.png
VIM PB probe
- SOD1-PA.png
SOD1 PA probe
- SOD1-PB.png
SOD1 PB probe
- C-Probe Hybridization
- Using same cells as yesterday cells to a new tube such that there are 100000 cells present
- Human-256 uL; Mouse-85 uL
- Pellet cells via centrifugation at 600g for 3 min
- Combine 1.5 uL of each C probe into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to CPH buffer for final concentration of 100 nM (Add 2 uL probe mixture to 0.5mL hybridization buffer)
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
- Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
- Latch and Padlock Hybridization
- Pellet at 600xg for 3 min and remove supernatant
- Resuspend pellet in 100 uL LPH buffer; use Latch0001 and Padlock0001-68
- Incubate for 30 min at 37C following the sample matrix
- Wash twice with 200 uL wash buffer
- Wash by pelleting cells at 600xg for 3 min
- Resuspend with 20 uL 1x Amp Ligase buffer
- Circularization
- Prepare 20 uL KLN mix
- Add 2 uL KLN mix to 20 uL of reaction
- Incubate at 55C overnight (XX hours)
- Continued tomorrow
Sample | Lane | Batch | AmpR Index | Cells | 2X Kapa SYBR qPCR MM | 100 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | A1 | B1 | 1 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 2 | A2 | B2 | 2 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 3 | A3 | B3 | 3 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 4 | A4 | B4 | 4 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 5 | A5 | B5 | 5 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 6 | A6 | B6 | 6 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
Sample 7 | A7 | B7 | 7 | 2 | 25 | 0.5 | 5 | 17.5 | 50 |
qPCR Results
C Probe SOD1/VIM Test
On Andrew's advice I'm also going to do another test using mouse cells (and human cells), this time with C-probes. To test more than one variable, I'm also going to use SOD1 and VIM. Below I've included the BLAST results testing the human probes against the mouse genome and transcriptome. VIM PA actually has a near-perfect match, but VIM PB does not match anywhere near VIM PA, so both probes should be unable to bind.
Protocol
Buffer Prep
Stringent Wash Buffer
Reagent | Stock | Final | Dilution | Amt in 1 mL (uL) |
PBS | 10X | 1X | 10 | 100 uL |
SSC | 20X | 4X | 5 | 200 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 10 uL |
nf H2O | NA | NA | NA | 690 uL |
LPH Buffer
Reagent | Stock | Final | Dilution | Amt in 100 uL (uL) |
Oligos | 10 uM | 100 nM | 100 | 1 uL |
SSC | 20X | 1X | 5 | 20 uL |
Rnasin | 40000 U/mL | 40 U/mL | 100 | 1 uL |
PBS | 10X | 1X | 10 | 10 uL |
nf H2O | NA | NA | NA | 68 uL |
Resuspended SOD1 C probes in 40 uL nfH2O each (100 uM)