Miniprep Plasmid DNA for Bio113 - Revision history

Macampbell at 00:59, 7 November 2018

2018-11-07T00:59:05Z

Macampbell at 18:37, 6 November 2018

2018-11-06T18:37:23Z

Macampbell at 18:27, 6 November 2018

2018-11-06T18:27:53Z

Macampbell at 19:28, 9 November 2017

2017-11-09T19:28:15Z

Macampbell: Created page with "'''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 aerate..."

2017-07-18T21:04:58Z

Created page with "'''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 aerate..."

New page

'''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'''
# Add 600 µL of O/N culture to an appropriately labeled 1.5 mL microfuge tube. 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°).
# While your samples are spinning, prepare Zymo-Spin II column (with white binding resin) by inserting into 2 mL collection tube. Be sure to label the spin column and the collection tube. Also label a second 1.5 mL microfuge tube for the final elution.
# 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 (from step 5 above). '''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.

@@ Line 3: / Line 3: @@
 '''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 with it.
 #Add 600 µL of O/N culture to an appropriately labeled 1.5 mL microfuge tube. 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.

@@ Line 3: / Line 3: @@
 '''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 with it.
+# 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 an appropriately labeled 1.5 mL microfuge tube. Save the rest of the O/N culture and keep them sterile.
+#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°.
@@ Line 14: / Line 13: @@
 # 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 (from step 5 above). '''Cut the lid off this tube before microfuging!'''
+# 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.