Lab Protocols

Question 1

What are the steps of mutPCR

Answer 1

1. typical Q5 PCR reaction with primers that have the corresponding mutation to be introduced
2. Screen PCR product with gel
3. 1uL of PCR product goes into a KLD reaction
4. 5ul of KLD product goes into NEB alpha cells for transformation
5. miniPrep -> plasmid DNA with mutation!!!

Question 2

Describe KLD protocol

Answer 2

1. Combine 1uL of Q5 PCR product with 3uL of MQ water, 5uL of KLD buffer and 1uL and 1uL of KLD enzyme mix
2. Mix throoughly up and down and incubate for 5 min
3. Put on ice or -20

Question 3

Describe transformation process

Answer 3

1. Go to -80deg area to take cells
2. Thaw them for 10 min.
3. Add ligated plasmid/KLD product (1-5uL), stir with tip ~5 times or so and flicker ~4 times.
4. Incubate on ice for 30 min.
5. set heater to 42 deg.
6. heat shock cells for 30 s exactly
7. Put on ice for 5 min and prepare SOC
8. add 950 uL SOC
9. Incubate for 1h at 37deg
10. Prepare plates (pour warm agarose with 1:1000 Amp)
11. Spread 25, 100 and rest (spinned down) uL of cells on plates and incubate overnight at 37