Difference between revisions of "Miniprep Plasmid DNA for Bio113"
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'''Lab Day''' | '''Lab Day''' | ||
| − | # Label two 1.5 mL tubes with your initials and the clone you will miniprep (e.g., X1). | + | # Label two 1.5 mL tubes with your initials and the clone you will miniprep (e.g., X1). Also, label one 2 mL collection tube and the spin column (around the rim) that is inside the collection tube. |
| − | #Add 600 µL of O/N culture to | + | #Add 600 µL of O/N culture to one of the appropriately labeled 1.5 mL microfuge tubes. Save the rest of the O/N culture and keep them sterile. |
# Add 100 µL 7X Lysis Buffer (blue color). Mix by inverting the tube 4-10 times. Solution should become clear blue instead of opaque. Proceed to the next step within 3 minutes. | # Add 100 µL 7X Lysis Buffer (blue color). Mix by inverting the tube 4-10 times. Solution should become clear blue instead of opaque. Proceed to the next step within 3 minutes. | ||
# Add 350 µL of Neutralization Buffer (yellow color; RNase A already added) and mix by inverting the tube until the entire solution and precipitate is yellow. This buffer is stored at +4 ° C. | # Add 350 µL of Neutralization Buffer (yellow color; RNase A already added) and mix by inverting the tube until the entire solution and precipitate is yellow. This buffer is stored at +4 ° C. | ||
# Microfuge full speed for 2 minutes at room temperature (RT°). | # Microfuge full speed for 2 minutes at room temperature (RT°). | ||
| − | |||
# Transfer ~900 µL yellow supernatant to Zymo-Spin II column. Do not transfer any of the solid precipitate. Better to leave some clear yellow liquid behind than to get greedy and transfer some solid material. | # Transfer ~900 µL yellow supernatant to Zymo-Spin II column. Do not transfer any of the solid precipitate. Better to leave some clear yellow liquid behind than to get greedy and transfer some solid material. | ||
# Microfuge full speed for 2 minutes at RT°. | # Microfuge full speed for 2 minutes at RT°. | ||
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# Add 200 µL Endo-Wash Buffer to the Zymo-Spin II column. Spin full speed for 15 seconds at RT°. Repeat this step a second time. No need to empty flow through until second wash is completed. | # Add 200 µL Endo-Wash Buffer to the Zymo-Spin II column. Spin full speed for 15 seconds at RT°. Repeat this step a second time. No need to empty flow through until second wash is completed. | ||
# Add 400 µL Zyppy Wash Buffer (with ethonol already added). Spin full speed for 30 seconds at RT°. Discard liquid flowthrough. Repeat this step a second time. Discard liquid flowthrough and and the 2 mL collection tube after the second wash. The DNA is still in the spin column. | # Add 400 µL Zyppy Wash Buffer (with ethonol already added). Spin full speed for 30 seconds at RT°. Discard liquid flowthrough. Repeat this step a second time. Discard liquid flowthrough and and the 2 mL collection tube after the second wash. The DNA is still in the spin column. | ||
| − | # Transfer Zymo-Spin II column to a clean and appropriately labeled 1.5 mL microfuge tube | + | # Transfer Zymo-Spin II column to a clean and appropriately labeled 1.5 mL microfuge tube. '''Cut the lid off this tube before microfuging!''' |
# Add 30 µL Zyppy Elution Buffer to the center of the to the Zymo-Spin II column. Let it stand for 1 minute to maximize yield. | # Add 30 µL Zyppy Elution Buffer to the center of the to the Zymo-Spin II column. Let it stand for 1 minute to maximize yield. | ||
# Microfuge full speed for 30 seconds at RT°. SAVE THE LIQUID with your plasmid. Discard the spin column. | # Microfuge full speed for 30 seconds at RT°. SAVE THE LIQUID with your plasmid. Discard the spin column. | ||
# You can NanoDrop the DNA to determine the concentration. | # You can NanoDrop the DNA to determine the concentration. | ||
Latest revision as of 00:59, 7 November 2018
Day Before Lab Day (4:30 pm) For each miniprep, grow 2 mL of LB + ampicillin culture, 37° C, overnight (O/N); shake at 250 RPM to make sure the cultures are well aerated.
Lab Day
- Label two 1.5 mL tubes with your initials and the clone you will miniprep (e.g., X1). Also, label one 2 mL collection tube and the spin column (around the rim) that is inside the collection tube.
- Add 600 µL of O/N culture to one of the appropriately labeled 1.5 mL microfuge tubes. Save the rest of the O/N culture and keep them sterile.
- Add 100 µL 7X Lysis Buffer (blue color). Mix by inverting the tube 4-10 times. Solution should become clear blue instead of opaque. Proceed to the next step within 3 minutes.
- Add 350 µL of Neutralization Buffer (yellow color; RNase A already added) and mix by inverting the tube until the entire solution and precipitate is yellow. This buffer is stored at +4 ° C.
- Microfuge full speed for 2 minutes at room temperature (RT°).
- Transfer ~900 µL yellow supernatant to Zymo-Spin II column. Do not transfer any of the solid precipitate. Better to leave some clear yellow liquid behind than to get greedy and transfer some solid material.
- Microfuge full speed for 2 minutes at RT°.
- Discard liquid flowthrough and reinsert Zymo-Spin II column into same collection tube.
- Add 200 µL Endo-Wash Buffer to the Zymo-Spin II column. Spin full speed for 15 seconds at RT°. Repeat this step a second time. No need to empty flow through until second wash is completed.
- Add 400 µL Zyppy Wash Buffer (with ethonol already added). Spin full speed for 30 seconds at RT°. Discard liquid flowthrough. Repeat this step a second time. Discard liquid flowthrough and and the 2 mL collection tube after the second wash. The DNA is still in the spin column.
- Transfer Zymo-Spin II column to a clean and appropriately labeled 1.5 mL microfuge tube. Cut the lid off this tube before microfuging!
- Add 30 µL Zyppy Elution Buffer to the center of the to the Zymo-Spin II column. Let it stand for 1 minute to maximize yield.
- Microfuge full speed for 30 seconds at RT°. SAVE THE LIQUID with your plasmid. Discard the spin column.
- You can NanoDrop the DNA to determine the concentration.
