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Worm lysis and PCR

Lysis buffer – need 15uL per single worm sample - make fresh each time

10 uL 10X PCR buffer

1uL Proteinase K (10mg/mL) - in -20 freezer

89 uL sterile water

Worm lysis

1. prepare and label PCR strips or 96 well plate

2. place 15uL lysis buffer into each tube using repeater

3. pick one adult worms into drop

4. close tubes or plate

5. spin in benchtop centrifuge

6. freeze at -20 or -80 (optional)

7. place in thermal cycler

8. use program Worm DNA on AB thermal cycler (or “boil worm” on AB 2720)

65°C 1hr -> 95°C 30 min -> 4°C forever

PCR for FLAG::sygl-1 genotyping

20uL rxn

____rxns

Water

12.8 uL

10X Taq Buffer

2 uL

10mM dNTPs

.4 uL

10uM sygl-1 primer F ZK47

.4 uL

10uM sygl-1 primer R ZK48

.4 uL

Template (undiluted

single worm lysis)

4 uL

Taq

0.1 uL

Thermal Cycler Program -this sample one is named ZUZPCR on cycler named BUDDHA

95°C for   5min

95°C for  30sec denature  ---

58°C for  60sec anneal         |   35x

68°C for  30sec elongate   ---

68°C for    5min

12°C  hold                                                                    total time: ~2 hours, 30 minutes

Note: for lst-1::FLAG genotying, use the same anealing temp, but use primers ZK28 and ZK29.

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