Project 1

Week 1

Week 2

Protocols

PCR protocol

PCR program design

1. Initial denaturation for 2 minutes at 95⁰C.
2. Denature for 1 minute at 95⁰C.
3. Anneal primers for 30 seconds at temperature ~5⁰C below melting temperature of primers.
4. Extend DNA at 72⁰C using each 1-2 minutes per kilobase of product, depending on polymerase used (see manufacturer’s instructions).
5. Repeat steps 2-4 for 25-30 cycles.
6. Final extension for 10 min at 72⁰C

PCR product purification using NucleoSpin

1. Mix 1 volume of sample with 2 volumes of NT buffer in an 1,5 ml Eppendorf tube.
2. Place a column into a 2 ml collection tube and load the sample.
3. Centrifuge at 11.000 g for 1 min.
4. Discard flow through and place the column back into the collection tube.
5. Add 600 µl NT3 buffer and centrifuge at 11.000 g for 1 min.
6. Discard flow through and place the column back into the collection tube.
7. Centrifuge at 11.000 g for 2 min to remove NT3 buffer. Discard flow through.
8. Place the column into a clean 1,5 ml Eppendorf tube.
9. Add 30 µl of water or NE buffer and incubate for 1 min to increase the yield of eluted DNA.
10. Centrifuge at 11.000 g for 1 min.

Plasmid puriification

This purification is based on the “Zyppy Plasmid Miniprep Kit”

Amounts of bacterial culture: According to Zyppy the purification can be done on 600 ul cell culture, but our experience suggests that it is not enough for further processing/use of the DNA. We use 2-4 ml of cell culture.

1. Initial steps:
   • Add 1.5 ml of cell culture in LB medium to 2 ml eppendorf tube.
   • Spin at 15.000 g for 2 min.
   • Discard supernatant
   • Add 1.5 of cell culture (for a total of 3 ml cell culture)
   • Spin at 15.000 g for 2 min.
   • Remove as much supernatant as possible – pipette carefully. This is (the only) point of no return! To stop, freeze the pellet.
   • Add 600 ul of TE-buffer. Ensure that the pellet is completely suspended.
2. Add 100 ul 7x lysis buffer. Remember not to process more than 10 minipreps at a time.
3. Add 350 ul cold neutralization buffer. Mix gently and thoroughly (= all the way through ≠ violently)!
4. Spin at 15.000 g for 5 min.
5. Transfer the supernatant to the columns; be careful not to get some of the pellet! It’s better to leave some supernatant than to get some of the pellet. Several “lysis” can be poured together to up-concentrate.
6. Spin at 15.000 g for 30 sec.
7. Discard flow-through.
8. Add 200 ul endo-wash-buffer
   • Spin at 15.000 g for 30 sec.
9. Add 400 ul zyppy wash buffer
   • Spin at 15.000 g for 30 sec.
10. Transfer columns to clean 1.5 ml eppendorf tubes. Be careful when removing the tubes, the buffer may not touch the tip of the column! (if it happens, spin again).
11. Elute DNA in 30-100 ul of buffer of choice (TE/H2O/restriction buffer/Zyppy elution buffer). Add the buffer to the center of the column, but without touching the column material! If H2O, wait 5 min before proceeding to the final centrifugation step, as DNA is not easily suspended in water.
12. Spin at 15.000 g for 30 sec.
13. Check the purification by running a gel (at least until we get experienced with a high success rate).

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