IGEM:MIT/2007/Notebook/2007-6-12

Grad Advisors

 * Morning: HD
 * Afternoon: Eric

=LAB WORK=

Mini Prep

 * 1) Harvest the bacterial cells by centrifugation at 5000rpm for 5 min. at 4˚C
 * 2) Pipette/Decant out LB suspension
 * 3) Resuspend pelleted bacterial cells in 0.3 ml Buffer P1 (in Falcone fridge) and transfer to a microcentrifuge tube
 * 4) *ensure that RNase A has been added to Buffer P1
 * 5)  Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times (or until homogenous), and incubate at room temperature for (no more than) 5 min.
 * 6) * do not vortex
 * 7) * close Buffer P2 immediately after use
 * 8) Add 0.4 ml of chilled Buffer N3, mix immediately and thoroughly by vigorously inverting 4-6 times, and incubate (on ice) for 5 min.
 * 9) Centrifuge for 10min. at 13,000 rpm
 * 10) Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting.
 * 11) Centrifuge for 30-60 seconds on max speed. Discard flow-through
 * 12) Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.
 * 13) Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
 * 14) Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl water (or Buffer EB) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Nanodrop (~20ng/ul) Protocol

 * 1) Start ND-1000 program
 * 2) Select "nucleic acid"
 * 3) 2 µL water
 * 4) Push OK
 * 5) Wipe top and bottom
 * 6) 2 µL EB buffer (or water if used for elution)
 * 7) Select "blank"
 * 8) Wipe top and bottom
 * 9) 2 µl sample
 * 10) Select "measure"
 * 11) Record the content of DNA in the lower right hand corner in ng/µl

PCR amplification of EGFP

 * 1) Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
 * 2) Set Reaction Tubes/Plates on Ice
 * 3) Add the following components in a reaction vessel. Total volume should be between .5-20 µl.
 * 4) * 45 µl Platinum PCR SuperMix
 * 5) * Primers (200 nM final concentration per primer is recommended)
 * 6) * Template DNA solution
 * 7) Cap reaction vessel and load into a thermal cycler at 94°C
 * 8) Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
 * 9) Perform the following PCR amplification
 * 10) 94°C for 4 minutes
 * 11) 94°C for 1 minute
 * 12) 55°C for 1 minute
 * 13) 72°C for 1 minute
 * 14) repeat steps 2-4 35 times
 * 15) 72°C for 10 minutes
 * 16) 4°C forever

Obtaining a Cast

 * 1) got a PCR gel cast holder from supply room
 * 2) got PCR cast from neighboring bay
 * How to improvise a cast when you don’t have the cast holder (which makes two sides of the gel):
 * Use tape to make a wall for the cast
 * Seal edges with nail,
 * Don’t let tape hang over bottom or else cast will not sit flat.

Protocol for Preparing Agarose Gel

 * 1) Add 50 mL of 1% agarose solution to a 125 mL sterilized Erlenmeyer flask.
 * 2) *Pour relatively quickly so solution doesn’t cool and harden
 * 3) For every 50mL of gel, add 5 µL of CyberSafe DNA gel stain (10000X concentration).
 * 4) Swirl flask to mix gel stain and agarose solution
 * 5) *Gel stain is located on shelf above PCR machine
 * 6) Place 8-well comb into cast
 * 7) Pour solution into cast, until solution level nearly reaches the tops of the walls.
 * 8) *Gel will set in about 15-20 minutes.
 * 9) *You know it’s ready when gel is no longer clear
 * 10) You can get rid of bubbles and push hairs away from wells with a pipette tip
 * 11) Dispose of remaining agarose; either let it harden and shake it out into trash or pour it down the sink with a LOT of water.

Protocol for Preparing to Run a Gel Electrophoresis

 * 1) Combine for samples to be loaded into gel:
 * 2) *2 µL of sample/bp ladder
 * 3) *14 µL of water
 * 4) *4 µL of 5X loading buffer
 * 5) *Total volume: 20 µL
 * 6) Separated 2 uL of sample (into eppendorf strip) to run in the gel. Saved 48 µL of sample (in original vial).
 * 7) Flick vials to mix contents before you pipette them out
 * 8) Put 2 µL of 100bp ladder into 6th well
 * 9) Put 2 µL of 1kbp ladder into 7th well
 * 10) *PCR Tube Sequence: 1- 1+  2+  3-  3+   100bp   1kbp
 * 11) *Sample key (yesterday, the three groups PCR’d a sample w/ and w/o template):
 * 12) *;- :No template
 * 13) *;+ :Has template
 * 14) *;Number :Corresponds to team
 * 15) Put 1 mL of 5X loading buffer into an eppendorf tube (to avoid opening and reopening)
 * 16) *Loading buffer contains glycerol to make samples sink into wells and contains colored dye to color DNA
 * 17) *Yellow dye is light and runs with lightest bits to indicate when your sample is about to run off the end of the gel
 * 18) *NOTE: What does the 5X mean? It indicates the concentration of buffer (or whatever has that label) that should be in the final mixture. So in this case, Final Mixture = 20 µL and Concentration = 5X, so Volume of Buffer in Final Mixture = 20/5 = 4 µL
 * 19) Add 4 µL of loading buffer to each sample to run in the electrophoresis
 * 20) Add 14 µL of water to bring total volume of each vial to 20 µL

Protocol for Running a Gel Electrophoresis

 * 1) Load 18 µL per sample into the wells (these samples should be the ones you prepared in the above protocol)
 * 2) *Sample sequence in agarose gel wells:
 * 3) *Blank 1kbp  1-  1+  2+  3-  3+  .1kb
 * 4) *NOTE: 2+ lost some sample
 * 5) Run the electrophoresis at 85V for 45 minutes
 * 6) *If voltages is too high, your gel will melt (even though it will run more quickly)
 * 7) *The current puts an upper limit on voltage
 * 8) *Check on the gel periodically to make sure you haven’t run off the gel
 * 9) When electrophoresis is done, remove lid, lift mold with gel out of buffer, have paper towel ready to blot

PCR Cleanup

 * 1) Use QIAquick (a PCR cleanup kit made by QIAGEN):
 * 2) *NOTE: make sure ethanol is in Buffer PE mixture. We did not clean controls today
 * 3) Add five volumes of buffer PBI to one volume of PCR mix
 * 4) *We have 48 µL so add ~250 µL of buffer per sample
 * 5) *Not all of the 250 µL fit, so we put in 125 µL directly into sample vial and the other 125 µL into the QIAquick column
 * 6) Check that the color of the mixture is yellow (similar to Buffer PBI without the PCR sample).
 * 7) *If color is orange or violet, add 10 uL of 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
 * 8) Place a QIAquick spin column in a provided 2 mL collection tube
 * 9) To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 s
 * 10) Discard flow-through. Place QUIAquick column back into same tube.
 * 11) To wash, add .75 mL Buffer PE to the QIAquick column and centrifuge for 30-60s
 * 12) *if you let it sit for 1-2 min, reaction works better
 * 13) Discard flow-through and place the QIAquick column back in the same tube. Centrifuge for 1 min
 * 14) *NOTE: residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation
 * 15) Place QIAquick column in a clean 1.5 ml centrifuge tube
 * 16) To elute DNA, add 40 µL Buffer EB (10mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane.
 * 17) (The longer we let it sit there than the better it does.
 * 18) Centrifuge the column for 1 min.
 * 19) (Optional) For higher DNA concentration, add 30 uL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, then centrifuge.
 * 20) If purified DNA is to be analyzed on a gel, add 1 volume of loading dye to 5 volumes of purified DNA. Mix solution by pipetting up and down before loading the gel.

Notes for Double Digestion

 * Cut 2 places at once using 2 different enzymes.
 * With double digestion, must find compatible conditions for both restriction enzymes.
 * Use the NEB book and find Double enzyme chart.
 * XBaI and EcoR1.
 * BSA keeps things from sticking to each other.
 * So check the enzymes (look in Section 1) and see if require anything special.
 * If recommends BSA for only one, still use it.
 * Avoid Star activity-when restriction enzyme cuts when it’s not supposed to.
 * Happens when Glycerol is too high (above 5%). So most commercial enzymes are 50% glycerol.  Not frozen at -20 degrees.
 * Digestion rate of restriction enzymes: 1 µg of DNA/1hr/1 restriction enzyme
 * 1) Make digestion mixture for insert and vector (in separate vials).
 * 2) *IMPORTANT: ENZYME goes in LAST
 * 3) *Enzymes are located in big fridge next to centrifuge (now in our fridge?)
 * 4) *Our enzymes are in the BioBricks box
 * 5) *When pipetting enzymes, do it relatively quickly and don’t stick tip in too far, otherwise you will suck up more enzyme than you intended
 * 6) Place digestion mixtures into hot room for 3 hours

Components of Digestion Mixture:

 * Typical Components
 * DNA (NNX + inserts)
 * 10X Buffer (Buffer 2)
 * BSA (100x)
 * Enzymes (EcoR1 and XBa1)
 * Water (to get right dilution)
 * 25 µL digestion mixtures:
 * Mixture for the vector (NNX)
 * 5 µL = 1 µg DNA
 * 2.5 µL of NEB Buffer 2
 * 2.5 µL of BSA (because BSA is originally at 10X concentration)
 * .5 µL of EcoR1
 * .5 µL of Xba1
 * 14 µL of water
 * Mixture for single digestion (a control)
 * 5 µL DNA
 * 2.5 µL of NEB Buffer 2
 * 2.5 µL of BSA
 * .5 µL of EcoR1
 * 14.5 µL of water
 * Mixture for insert (EGFP)
 * Volume equivalent of 800 ng of DNA
 * 2.5 µL of Buffer
 * 2.5 µL of BSA
 * .5 µL of EcoR1
 * .5 µL of Xba1
 * Enough water to bring total volume to 25 µL

Protocol for Imaging Result and Gel Diagnostic
**Use NEB to find ladder images, check to see if your PCR results are where you expect them (near the correct bp band)
 * 1) “Gel Electrophoresis Imaging” *red light = measuring
 * 2) Remove gel from buffer solution (use paper towel)
 * 3) Place container with gel on slide (leave door open)
 * 4) *Light – “upper white”, Emission Filter – “short wave”, 500 ms
 * 5) *Get image and adjust to focus/center
 * 6) Close door
 * 7) *Change Light to “trans illumination”
 * 8) *Get image and adjust contrast to focus (re-image after each adjustment)
 * 9) Save to file – Endy  iGEM folders
 * 10) Export as .tif or .jpeg
 * 11) Print
 * 12) *Diagnostic:

Things we need

 * Medium latex gloves
 * timers
 * calculators