Stanford/BIOE44:Module 1:Day1: Difference between revisions
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#Apply 750uL of Buffer PE and centrifuge for 1 minute at max speed. | #Apply 750uL of Buffer PE and centrifuge for 1 minute at max speed. | ||
#Discard the flow through. Centrifuge the column for an additional 1 minute to remove residual wash buffer. | #Discard the flow through. Centrifuge the column for an additional 1 minute to remove residual wash buffer. | ||
#Place the column in a clean 1.5ml micro-centrifuge tube. Apply 50uL sterile water to the column. Let it stand for 1 minute. Centrifuge at max speed for 1 minute. | #Place the column in a clean 1.5ml micro-centrifuge tube. Apply 50uL sterile water to the column. Let it stand for 1 minute. Centrifuge at max speed for 1 minute. | ||
'''Measure the concentration'' | |||
#Measure the concentration of your newly prepped plasmid with the nano-drop. Record the concentration and 260/280 in your lab notebook. | |||
===Part 2: Restriction Enzyme Digest=== | ===Part 2: Restriction Enzyme Digest=== |
Revision as of 20:53, 30 March 2010
M1: Day 1 - Plasmid Extraction and Restriction Digest
Introduction
- overview of what we are going to do
- standard assembly
- plasmids
- parts
- promoters
- methods stuff:
- mini preps - purpose of each step
- restriction enzymes
- components of a digest
Extra Reading/References
- Standard Assembly Info
- Cambridge iGEM project 2009 - E. Chromi
- How are plasmids separated from chromosomal DNA?
In Class
Part 1: Plasmid extraction (aka miniprep)
At the beginning of class you will be given a 5mL culture of E. coli harboring a plasmid that contains a BioBrick part. We will extract the plasmid from the bacteria using a kit (QIAPrep Spin Miniprep kit). Then we'll measure the concentration of the plasmid DNA extracted using a nanodrop spectrophotometer.
Mini-Prep Protocol
Materials
- 5mL E. coli culture
- microcentrifuge tubes
- Buffers P1, P2, N3, and PE
- Qiaprep column
- sterile water
Collect the Cells
- Label three microcentrifuge tubes.
- Pour approx. 1.5 mL of culture into each tube.
- Centrifuge at 10000xg for 5 minutes.
- Aspirate off supernatant. Be care to not suck up your cell pellet.
Break the cells open
- Resuspend the cells in tube #1 with 250uL of Buffer P1.
- Transfer all liquid from tube #1 to tube #2 and resuspend the pellet.
- Repeat from tube #2 to tube #3. (Should end up with all the cells in tube #3. Throw away tubes 1 and 2)
- Add 250uL of Buffer P2 to cells. Invert the tube 4-6 times. Solution should become slightly clear and viscous. (This is the step where the cells are lysed and all the the proteins are denatured)
- Add 350uL of Buffer N3 to the solution. Mix immediately by inverting the tube 4-6 times. (Thick white goo will form - this is all the proteins precipitating out)
- Centrifuge for 10 minutes at max speed (approx 17000xg). While centrifuging, take out a column and label it.
Isolate the plasmids
- Apply the supernatant from the tube to the column by pipetting (approx 800ul). Be care to not pipette any of the white stuff - this will clog your column and reduce your yield.
- Centrifuge for 1 minute at max speed. Discard the flow through.
- Apply 750uL of Buffer PE and centrifuge for 1 minute at max speed.
- Discard the flow through. Centrifuge the column for an additional 1 minute to remove residual wash buffer.
- Place the column in a clean 1.5ml micro-centrifuge tube. Apply 50uL sterile water to the column. Let it stand for 1 minute. Centrifuge at max speed for 1 minute.
'Measure the concentration
- Measure the concentration of your newly prepped plasmid with the nano-drop. Record the concentration and 260/280 in your lab notebook.
Part 2: Restriction Enzyme Digest
We will take the plasmid we just extracted and set up a restriction digest to cut out the color generator genes.
For Next Time
- Please read: