====== Single Worm Lysis and PCR ======

 --- //[[madisonotero08@gmail.com|Madison Otero]] 2025/08/14 13:07//

===== Overview =====
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Purpose: to amplify regions of interest of DNA.


===== Materials=====
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^ PK Buffer        ^ PCR Master Mix  ^
| dH2O             | dH2O            |
| 20 mg/mL PK      | 10X Buffer      |
| 10X worm buffer  | Q solution      |
|                  | dNTPs           |
|                  | forward primer  |
|                  | reverse primer  |
|                  | Taq Polymerase  |

=====Procedure=====
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Before starting, check to see if there is dry ice in CRF 110.

**Part 1: Worm Lysis**
  - Make PK buffer using master mix listed in the notes.
  - Label lids with EtOH resistant marker (in drawer 49).
  - Add 10 uL of PK buffer to the lids of PCR tubes (10 uL per lid, one lid per worm).
  - Add one adult worm to each lid, put the tubes on the lids, and centrifuge.
  - Get dry ice from CRF 110 (small scoop is enough) and pour EtOH in the same container.
  - Put tubes with worms in the EtOH + dry ice mixture long enough for the liquid to freeze, then remove tubes until they are thawed. Repeat this for a total of three times. 
  - Put tubes in Thermocycler and use protocol "cook worm".
^ Time    ^ Temperature  ^
| 60 min  | 60 °C        |
| 15 min  | 95 °C        |
| ∞       | 10 °C        |

**Part 2: PCR**
  - Make master mix using recipe listed in the notes.
  - Vortex and centrifuge the master mix.
  - Label PCR tubes to distinguish strains and primer pairs, and copy this labeling plan in your notebook.
  - Add 10 uL of master mix into new PCR tubes - do NOT add DNA into the combined master mix. 
  - Add 2.5 uL DNA to PCR tubes that have 10 uL of master mix, the total should be 12.5 uL. 
  - Centrifuge the tubes in the mini centrifuge to mix the DNA into the master mix. 
  - Put the PCR tubes in the Thermocycler and use protocol "worm PCR".
  - Make sure the volume is 12.5 uL (can round to 13 uL if machine doesn't allow decimals), and edit settings to your need.
^ Step                  ^ Temperature  ^ Time           ^ Repetition  ^ Editing                                                 ^
| Initial Denaturation  | 95 °C        | 3 min          | x1          |                                                         |
| Denaturation          | 95 °C        | 1 min          | x40         |                                                         |
| Primer anneal         | 50-70 °C     | 1 min          | x40         | edit temperature based on specific primer Tm            |
| Extension             | 72 °C        | 30s - 2.5 min  | x40         | edit time based on length of product (~2-4 kb per min)  |
| Final Extension       | 72 °C        | 10 min         | x1          |                                                         |
| Hold                  | 4 °C         | ∞              | x1          |                                                         |

=====Notes=====
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**Recipe for PK Buffer**
^ Reagent             ^ Amount per Rxn  ^
| dH2O                | 8.5 uL          |
| 20 mg/mL PK buffer  | 0.5 uL          |
| 10X Worm Buffer     | 1 uL            |

**Recipe for PCR Master Mix**
^ Reagent         ^ Amount per Rxn  ^
| dH2O            | 5.375 uL        |
| 10X Buffer      | 1.25 uL         |
| Q solution      | 2.5 uL          |
| dNTP            | 0.25 uL         |
| forward primer  | 0.25 uL         |
| reverse primer  | 0.25 uL         |
| Taq Polymerase  | 0.125 uL        |
| DNA             | 2.5 uL          |

  * Multiply recipe by how many reactions you need plus 1-3 extra to account for pipetting error (i.e. 10 worm -> multiply recipe by 12).
  * For PCR master mix, do not multiply DNA as part of the master mix calculation. Make the master mix without DNA, add it to all the tubes, then add DNA into each tube. 
  * If dry ice is not available, flash freezing the worms can be done with liquid nitrogen, but ensure you have a way of getting it before starting (do not have any currently in lab as of 09/05/2025).