Daniel:Notebook/PosSequencing/2017-5-22

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Revision as of 18:55, 22 May 2017 by >Djacobse (→‎Protocol-Part 2)
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Single Stranded Probe Binding Test (Started Friday May 19)

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Protocol (Continued from Saturday May 20)

  1. Dye hybridization
    1. Mix 20 uL ssDNA (lambda digested) and 20 uL dsDNA (original, non-digested template)
    2. Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
    3. Incubate in the dark (RT) for 15 minutes
    4. Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
    5. Add 15 uL to a slide for analysis
    6. Seal coverslip with nail polish and wait approximately 30 minutes to dry
  2. Imaging Parameters
    1. Confocal Microscope
    2. 488 Laser-3.00%
      1. PMT Trans-Gain 225V
      2. PMT1-Range:500-550-Gain 550V
      3. PMT3-Range:750-800-Gain 600V
    3. 642 Laser-8.00%
      1. PMT Trans-Gain 300V
      2. PMT1-Range:500-550-Gain 6000V
      3. PMT3-Range:750-800-Gain 550V

Results

Raw Images

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
  1. Bead binding
    1. Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)
    4. Add 5 uL barcode2 oligo (300 nM) and 10 uL nfH2O; incubate at RT for 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant

pH Denaturation Samples(1-4)

  1. pH denaturation
    1. Resuspend beads in 50 uL of appropriate pH solution (see sample matrix)
    2. Incubate 2 minutes RT
    3. Magnet pulldown 1 min; remove supernatant
    4. Resuspend in 50 uL 1X TE buffer

Heat Denaturation Samples (5-8)

  1. Heat denaturation
    1. Resuspend beads in 50 uL of TE-TritonX 100 0.1% (TE-T)
    2. Incubate 3 minutes at the appropriate temperature according to sample matrix
    3. Magnet pulldown 1 min; remove supernatant
    4. Resuspend in 50 uL 1X TE buffer

Lambda Exonuclease Sample (9)

  1. Phosphorylation
    1. Resuspend beads in solution using the following table
    2. 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
    3. Incubate at 37C for 30 minutes
    4. Incubate at 65C for 20 minutes
  2. Lambda Exonuclease Digestion
    1. Magnet pulldown 1 minute and remove supernatant
    2. Resuspend in solution according to following table
    3. Reagent Final Conc. uL Added
      Reaction Buffer 10X 1X 5
      Lamda Exonuclease (10 U/uL) 2 U/uL 2
      nfH2O NA 43
      Total   50
    4. Incubate at 37C for 30 minutes
    5. Add 2 uL 0.5M EDTA and mix well; incubate at RT for 5 minutes
    6. Magnet pulldown and resuspend in 50 uL 1X TE buffer

Protocol-Part 2

  1. Single primer qPCR
    1. Make the following master mixes
    2. 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
    3. Add 35 uL appropriate master mix to the lanes according to plate layout
    4. Add 5 uL samples according to plate layout
    5. [[Image:|400px]]
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold