Revision as of 18:25, 6 March 2017 by >Djacobse
Padlock0601 Test (Started Yesterday)
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Protocol-Part 3
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Column Purification-Qiaquick
- Add 150 uL (5X) PB (binding buffer) to 30 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
- Repeat wash step;
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Elute with 30 uL EB
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification
- Samples will be aliquoted under following rules
Samples
|
Condition
|
Sample X (1-4, AB)
|
Unpurified
|
Sample Y (1-4, AB)
|
Purified
|
A Samples (1-4)
|
Normal Primer
|
B Samples (1-4)
|
Phosphorothiorate Primer
|
- Prepare 8.2X Master mix according to table below
Reagent
|
Single Rxn Vol
|
16.2X MM uL
|
Template
|
4
|
0
|
RCA Primer (10 uM)
|
2.5
|
0
|
dNTP (1 mM)
|
5
|
81
|
10X Buffer
|
2
|
32.4
|
Phi29
|
1
|
0
|
BSA (10 mg/mL)
|
0.4
|
6.48
|
nfH2O
|
5.1
|
82.62
|
Total
|
20
|
202.5
|
- Add 12.5 uL master mix to each tube
- Add 4 uL template according to table
- Add 2.5 uL correct primer according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 3 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Protocol-Part 4
- BglII Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 4.2X master mix
Reagent
|
Single Rxn Vol
|
8.2X MM uL
|
RCA Reaction Product
|
5
|
0
|
Bglii Cutter Primer (10 uM)
|
2
|
16.4
|
10X Buffer 3.1
|
2
|
16.4
|
Bglii
|
1
|
0
|
nfH2O
|
10
|
82
|
Total
|
20
|
114.8
|
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make 25.2X qPCR master mix according to following recipe
- 529.2 uL nfH2O
- 630 uL SYBR Fast
- 25.2 uL 10 uM AmpF
- 25.2 uL 10 uM AmpR-Ind20
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout
File:PlateLayout-20170302-Padlock0601Test.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
qPCR for post-Bglii digestion and gel images for all data continued tomorrow