Daniel:Notebook/PosSequencing/2017-5-22
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Single Stranded Probe Binding Test (Started Friday May 19)
Protocol (Continued from Saturday May 20)
- Dye hybridization
- Mix 20 uL ssDNA (lambda digested) and 20 uL dsDNA (original, non-digested template)
- Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
- Incubate in the dark (RT) for 15 minutes
- Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
- Add 15 uL to a slide for analysis
- Seal coverslip with nail polish and wait approximately 30 minutes to dry
- Imaging Parameters
- Confocal Microscope
- 488 Laser-3.00%
- PMT Trans-Gain 225V
- PMT1-Range:500-550-Gain 550V
- PMT3-Range:750-800-Gain 600V
- 642 Laser-8.00%
- PMT Trans-Gain 300V
- PMT1-Range:500-550-Gain 6000V
- PMT3-Range:750-800-Gain 550V
Results
Raw Images
- 20170522-LambdaExonucleaseDigestion.lif Sample1-Field1-SNAP ch00.jpg
Sample 1 Field 1
- 20170522-LambdaExonucleaseDigestion.lif Sample1-Field2-SNAP ch00.jpg
Sample 1 Field 2
- 20170522-LambdaExonucleaseDigestion.lif Sample1-Field3-SNAP ch00.jpg
Sample 1 Field 3
- 20170522-LambdaExonucleaseDigestion.lif Sample1-Field4-Cy5-SNAP ch00.jpg
Sample 1 Field 4-Cy5
- 20170522-LambdaExonucleaseDigestion.lif Sample2-Field1-SNAP ch00.jpg
Sample 2 Field 1
- 20170522-LambdaExonucleaseDigestion.lif Sample2-Field2-SNAP ch00.jpg
Sample 2 Field 2
- 20170522-LambdaExonucleaseDigestion.lif Sample2-Field3-SNAP ch00.jpg
Sample 2 Field 3
- 20170522-LambdaExonucleaseDigestion.lif Sample2-Field3-Cy5-SNAP ch00.jpg
Sample 2 Field 3-A488
Counts
Denatuation Test
I'm going to test across the board for optimal denaturation options. The key to doing denaturation is to denature the double stranded DNA without breaking the streptavidin-biotin bond. This means I'll have to do bead binding first. The principal three options for denaturing the double stranded DNA are:
- Heat; 95.0C is the most common but by no means the only; 95C 3 minutes was the given temp but
- pH-Alkaline pH is a good choice for denaturation; DNA seems to denature around pH 10 and biotin-Strep around 11.0, so wiggle room?
- Lambda exonuclease-Least palatable but most effective; LXO will chew up the second strand, as shown above, but it means enzyme digestion after every amplification, which isn't ideal.
Rather than worrying about which one to test, we're going wholesale! I'll use qPCR again as the tester, since that's just a waste of dye probes otherwise.
Protocol
Sample Matrix
Sample No | Variable | Condition |
1 | pH | pH=9.0 |
2 | pH | pH=10.0 |
3 | pH | pH=11.0 |
4 | pH | ph=12.0 |
5 | Heat | 95.0C (3 min) |
6 | Heat | 90.0C (3 min) |
7 | Heat | 85.0C (3 min) |
8 | Heat | 80.0C (3 min) |
9 | LXO (++++) | Positive Control-Lambda Exonuclease digestion |
10 | dsDNA (----) | Negative Control-Nothing |
- Bead binding
- Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)
- Add 15 uL barcode2 oligo; incubate at RT for 15 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
pH Denaturation Samples(1-4)
- pH denaturation
- Resuspend beads in 50 uL of appropriate pH solution (see sample matrix)
- Incubate 2 minutes RT
- Magnet pulldown 1 min; remove supernatant
- Resuspend in 50 uL 1X TE buffer
Heat Denaturation Samples (5-8)
- Heat denaturation
- Resuspend beads in 50 uL of TE-TritonX 100 0.1% (TE-T)
- Incubate 3 minutes at the appropriate temperature according to sample matrix
- Magnet pulldown 1 min; remove supernatant
- Resuspend in 50 uL 1X TE buffer
Lambda Exonuclease Sample (9)
- Phosphorylation
- Resuspend beads in solution using the following table
- Incubate at 37C for 30 minutes
- Incubate at 65C for 20 minutes
- Lambda Exonuclease Digestion
- Prepare 2 reactions according to following table
- Incubate at 37C for 30 minutes
- Incubate at 80C for 10 minutes
- Hold at 12C
Reagent | Final Conc. | uL Added |
Reaction Buffer 10X | 1X | 5 |
ATP (10 mM) | 1 mM | 5 |
T4 PNK (10 U/uL) | 10 Units | 1 |
nfH2O | NA | 39 |
Total | 50 |
Reagent | Final Conc. | uL Added |
Phosphorylated Mix | (<2 ug) | 20 |
Reaction Buffer 10X | 1X | 5 |
Lamda Exonuclease (10 U/uL) | 2 U/uL | 2 |
nfH2O | NA | 23 |
Total | 50 |
Protocol-Part 2
- Single primer qPCR
- Make the following master mixes
- Add 37 uL or 38 uL (dual) to the appropriate lanes
- Add 2 uL samples and 1 (or 2) uL primer according to plate layout File:PlateLayout-20170520-SinglePrimer.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
Reagent | uL in Single Rxn | Master Mix P2 (3.2X) | Master Mix BioU (3.2X) | Master Mix Dual (2.2X) |
Primer 2 (10 uM) | 1 | 3.2 | 3.2 | 2.2 |
BioU Primer (10 uM) | 1 | 0 | 0 | 2.2 |
2X Kapa SYBR Master Mix | 20 | 64 | 64 | 44 |
nfH2O | 17 | 54.4 | 54.4 | 35.2 |
Total | 121.6 | 121.6 | 83.6 |