Daniel:Notebook/PosSequencing/2017-5-22

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Revision as of 18:01, 22 May 2017 by >Djacobse (→‎Denatuation Test)
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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 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