DNA Extraction using Promega DNA Kit_28017905

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title: "TheNessLab : DNA Extraction using Promega DNA Kit"

1. TheNessLab
2. The Ness Lab Wiki
3. Chlamydomonas information and protocols

TheNessLab : DNA Extraction using Promega DNA Kit

Created by Wen Chen on Nov 26, 2018

Please follow the instructions below to extract DNA using Promega DNA kit.

Materials:

• Liquid nitrogen
• 2 mL microcentrifuge tubes
• Tongs
• Metallic beads
• Styrofoam box with ice
• Microcentrifuge
• Promega DNA kit
• Homogenizer
• Promega Maxwell RSC machine

Procedure:

Before you start, make sure no one is using Promega Maxwell RSC machine.

1. Spin culture down to collect pellet in a 2 mL microtube.
2. Wear proper PPE for liquid nitrogen.
3. Dip the tube in liquid nitrogen briefly using tongs until it stops bubbling to flash freeze the pellet. 
4. Place the tube on ice.
5. Add 300 uL TLA to the tube.
6. (Optional) Add 10 uL RNAase.
7. Add 3 metallic beads.
8. Put tube in a homogenizer. Shake at 25 shakes/second for 30 seconds.
   • Edit the parameter to 25 shakes/s. Then press "lock" and "start."
   • If you are using Sasha's homogenizer, try to organize the tubes so that they are balanced.
9. Spin tubes at 13000 rcf for 2 min in a microcentrifuge. Collect supernatant.
10. Turn on Promega Maxwell RSC machine.
11. Click on "Maxwell RSC"→ "Start"→ "Plant DNA AS1490"→"Proceed."
12. Select slots where you'll be putting samples. If there are less than 6 samples, center the samples in the middle.
13. Take the numbered black tray out.
14. Insert cartridges with numbers facing away from numbers.
15. Remove film on cartridges.
16. Place 1 elution tube in each tube slot.
17. Add 50 uL nuclease-free water supplied with the Promega DNA extraction kit. 
18. Add 300 uL nuclease-free water to Well 1 (the cartridge well farthest away from the tube slots).
    • Do not cross contaminate the liquid in Well 1 with nuclease-free water.
19.  Add 300 uL supernatant in Well 1. 
20. Place one plunger in Well 8.

• If your DNA extract turns grey, ask Safa what to do.
• The Promega DNA kit also comes with a manual if I didn't explain the steps clear enough.

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