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
   • ensure that RNase A has been added to Buffer P1
4. 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.
   • do not vortex
   • close Buffer P2 immediately after use
5. 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.
6. Centrifuge for 10min. at 13,000 rpm
7. Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting.
8. Centrifuge for 30-60 seconds on max speed. Discard flow-through
9. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.
10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
11. 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.
   • 45 µl Platinum PCR SuperMix
   • Primers (200 nM final concentration per primer is recommended)
   • Template DNA solution
4. Cap reaction vessel and load into a thermal cycler at 94°C
5. Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
6. Perform the following PCR amplification
   1. 94°C for 4 minutes
   2. 94°C for 1 minute
   3. 55°C for 1 minute
   4. 72°C for 1 minute
   5. repeat steps 2-4 35 times
   6. 72°C for 10 minutes
   7. 4°C forever

Run agarose gel of PCR products

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):
  1. Use tape to make a wall for the cast
  2. 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.
   • Pour relatively quickly so solution doesn’t cool and harden
2. For every 50mL of gel, add 5 µL of CyberSafe DNA gel stain (10000X concentration).
3. Swirl flask to mix gel stain and agarose solution
   • Gel stain is located on shelf above PCR machine
4. Place 8-well comb into cast
5. Pour solution into cast, until solution level nearly reaches the tops of the walls.
   • Gel will set in about 15-20 minutes.
   • You know it’s ready when gel is no longer clear
6. You can get rid of bubbles and push hairs away from wells with a pipette tip
7. 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 µL of sample/bp ladder
   • 14 µL of water
   • 4 µL of 5X loading buffer
   • Total volume: 20 µL
2. Separated 2 uL of sample (into eppendorf strip) to run in the gel. Saved 48 µL of sample (in original vial).
3. Flick vials to mix contents before you pipette them out
4. Put 2 µL of 100bp ladder into 6th well
5. Put 2 µL of 1kbp ladder into 7th well
   • PCR Tube Sequence: 1- 1+ 2+ 3- 3+ 100bp 1kbp
   • Sample key (yesterday, the three groups PCR’d a sample w/ and w/o template):

     -

     No template

     +

     Has template

     Number

     Corresponds to team

6. Put 1 mL of 5X loading buffer into an eppendorf tube (to avoid opening and reopening)
   • Loading buffer contains glycerol to make samples sink into wells and contains colored dye to color DNA
   • Yellow dye is light and runs with lightest bits to indicate when your sample is about to run off the end of the gel
   • 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
7. Add 4 µL of loading buffer to each sample to run in the electrophoresis
8. 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)
   • Sample sequence in agarose gel wells:
   • Blank 1kbp 1- 1+ 2+ 3- 3+ .1kb
   • NOTE: 2+ lost some sample
2. Run the electrophoresis at 85V for 45 minutes
   • If voltages is too high, your gel will melt (even though it will run more quickly)
   • The current puts an upper limit on voltage
   • Check on the gel periodically to make sure you haven’t run off the gel
3. 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):
   • NOTE: make sure ethanol is in Buffer PE mixture. We did not clean controls today
2. Add five volumes of buffer PBI to one volume of PCR mix
   • We have 48 µL so add ~250 µL of buffer per sample
   • 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
3. Check that the color of the mixture is yellow (similar to Buffer PBI without the PCR sample).
   • 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
4. Place a QIAquick spin column in a provided 2 mL collection tube
5. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 s
6. Discard flow-through. Place QUIAquick column back into same tube.
7. To wash, add .75 mL Buffer PE to the QIAquick column and centrifuge for 30-60s
   • if you let it sit for 1-2 min, reaction works better
8. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge for 1 min
   • NOTE: residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation
9. Place QIAquick column in a clean 1.5 ml centrifuge tube
10. To elute DNA, add 40 µL Buffer EB (10mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane.
11. (The longer we let it sit there than the better it does.
12. Centrifuge the column for 1 min.
13. (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.
14. 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).
   • IMPORTANT: ENZYME goes in LAST
   • Enzymes are located in big fridge next to centrifuge (now in our fridge?)
   • Our enzymes are in the BioBricks box
   • 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
2. 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

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)
   • Light – “upper white”, Emission Filter – “short wave”, 500 ms
   • Get image and adjust to focus/center
4. Close door
   • Change Light to “trans illumination”
   • Get image and adjust contrast to focus (re-image after each adjustment)
5. Save to file – Endy  iGEM folders
6. Export as .tif or .jpeg
7. Print
   • Diagnostic:

**Use NEB to find ladder images, check to see if your PCR results are where you expect them (near the correct bp band)

Digest vector and PCR product

Figure 1: Gel Image

Things we need

• Medium latex gloves
• timers
• calculators