Team:KULeuven/Protocols

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Protocols Wet Lab

• Miniprep (Qiagen)
• ...

Plasmid DNA purification

Materials

• LB broth with appropriate antibiotic
• 15 ml tube
• incubation oven at 37°C
• microcentifuge tubes
• table-top microcentrifuge
• buffer P1 (Qiagen kit)
• buffer P2 (Qiagen kit)
• buffer N3 (Qiagen kit)
• QIAprep spin columns
• transparant tape
• buffer PE (Qiagen kit)
• buffer EB (Qiagen kit)
• nanodrop

Procedure

1. Inoculate a single colony into 5 ml of LB with the appropriate antibiotic and incubate at 37°C for 12-16 hours (liquid culture).
2. Put 1.5 ml of this liquid culture in a microcentrifuge tube and centrifuge at 8500 rpm for 3 minutes at room temperature.
3. Remove all traces of supernatant by inverting the tube.
4. Resuspend the pelleted cells in 250 $\mu$l Buffer P1 and vortex until no cell clumps remain.
5. Add 250 ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times. !! Do not vortex and do not allow the reaction to proceed for more than 5 minutes !
6. Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The solution will become cloudy.
7. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will form.
8. Apply the supernatant of step 4 to a QIAprep spin column by decanting. When you label the columns, cover this label with tape (the ink tends to dissolve).
9. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through. If you made several tubes of one sample, you can repeat the steps 8 and 9 in the same QIAprep spin column. This way, the concentration of plasmid DNA will be higher.
10. Wash the QIAprep spin column with 0.75 ml Buffer PE. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through !!
11. Centrifuge for an additional minute to remove all residual wash buffer.
12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 ul Buffer EB to the centre of the QIAprep spin column, let stand for 1 minute and centrifuge for 1 minute at 13000 rpm.
13. Measure the concentration of plasmid DNA with the nanodrop (ng/ul). Use Buffer EB as blank.

Restriction Digest

Materials

• restriction enzymes ( EcoRI, SpeI, PstI and XbaI)
• restriction buffer H
• milliQ
• plasmid DNA
• blue juice
• Smart ladder (reference)

Procedure

Here we describe a 20 ul reaction. The used restriction enzymes are from Roche. Prepare several tubes of the same sample.

• If you want to digest with a mixture of EcoRI and SpeI, add the following to a microcentrifuge tube:
  • 500 ng plasmid DNA
  • (14-X) ul milliQ
  • 2 ul buffer H: Vortex buffer before pipetting to ensure that it is well-mixed.
  • 1 ul EcoRI and 1 ul SpeI. Vortex enzyme before pipetting to ensure that it is well-mixed. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 ul, just touch your tip to the surface of the liquid when pipetting. The restriction enzymes must be the last thing you add. Allways keep them on ice.
• If you want to digest with a mixture of EcoRI and XbaI, add the following to a microcentrifuge tube:
  • 500 ng plasmid DNA
  • (17-X) ul milliQ
  • 2 ul buffer H: Vortex buffer before pipetting to ensure that it is well-mixed.
  • 1 ul XbaI: Vortex enzyme before pipetting to ensure that it is well-mixed. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 ul, just touch your tip to the surface of the liquid when pipetting. The restriction enzymes must be the last things you add. Always keep them on ice.

1. Incubate the tubes at 37°C for 1.5-2 hours (heat block or oven). In the mean time, you can prepare the agarose gel.
2. For the digest with XbaI: put 2 ul of DNA with 8 ul milliQ and 2 ul BlueJuice on gel to check whether the enzym has properly cut. Then, add 1ul EcoRI to the rest of the mixture and incubate for 1.5-2 hours at 37°C.
3. Add 4 ul BlueJuice to each tube.
4. Load 5 ul reference mixture (ladder) onto the gel.
5. Load 25 ul digest onto the gel. Make sure that you have multiple lanes with the same BioBrick (higher concentration).
6. Start the electrophoresis (this should take about 1 hour). Look at the result under UV-light and cut out the correct fragment.
7. Purify the obtained fragment.

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