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.