Team:iTesla-SoundBio/DigestPurificationGelandGelExtractionProtocolTemplate

Highlighted text are things that you need to fill in or change for your particular lab notebook depending on the variables and concentrations.

 

Digesting Fragment 1 with EcoRI and PstI

 

Note: All steps in this process are very rough on the DNA. This includes being surrounded by enzymes, being in LD, being in a gel, and being in buffers. Therefore, all steps in this process should be completed as quickly and efficiently. Don’t rush, but don’t go out for ice cream in between steps (did that once sorry).

 

Materials:

-Optimized Buffer (1.1, 2.1, or 3.1) [Sniffles, “Sound Bio Enzymes Box on lower shelf]

-DNA to digest [           ]

-Enzymes [          ]

-RNase free water [SoundBio Storage, on wooden shelf aliquoted microcentrifuge tubes]

 

Procedure:

  1. Label pcr tube/s  __________
  2. Add the following into the pcr tubes:
    1. Determine optimized buffer via <a style="text-decoration: none;" href="https://nebcloner.neb.com/#!/redigest">https://nebcloner.neb.com/#!/redigest</a>
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Digest (40ul total)

-____ ul H2O (to a 40 ul or 50 ul reaction)

-4 ul NEB buffer

-1 ug of DNA to be Digested

-1 ul Enzyme 1

-1 ul Enzyme 2

***The enzymes are very temperature sensitive, so add those quickly and at the very end!***

  1. After incubation, heat inactivate the enzymes by running a thermocycling program that is 37 C for 1 hour, and then the highest heat inactivation for 20 mins. Determine your enzymes heat inactivation temps here: <a style="text-decoration: none;" href="https://www.neb.com/tools-and-resources/usage-guidelines/heat-inactivation">https://www.neb.com/tools-and-resources/usage-guidelines/heat-inactivation</a>

 

Gel Purification:

 

Gel Purification Materials:

  • TAE solution [carboy near the sink]
  • 250 mL flask
  • Agarose [Falcon tube on top of gel cart]
  • Microwave
  • Digested ________ [from last step]
  • 1 kb ladder  [Blue Citizen Salmon box located on top shelf of Sniffles]
  • 5X Loading Dye [gel cart in bag]
  • 20,000X Gel Stain [foil envelope in door of fridge]
  • Parafilm [next to microwave]
  • Scale and weigh boat
  • Graduated cylinder
  • Gel comb (ten wells), tray, etc.
  • Power supply [on top of microwave]
  • Razor

 

Procedure:

  1. Fill flask with 60 mL of TAE using graduated cylinder
  2. Weigh out 0.6 g of agarose and add it to TAE
  3. Microwave TAE solution until its clear and agarose is entirely dissolved. I started with 30 seconds and did 10 second increments after.
  4. Once TAE solution is sufficiently cooled, add 3 uL of 20,000X gel stain and swirl it around a bit.
  5. Pour TAE solution into gel tray. Make sure it’s taped around the sides.
  6. Once the solution has solidified, then carefully remove comb.
  7. Fill gel box with enough TAE to cover the gel. Make sure the gel tray rests perfectly in the middle of the gel box. Get rid of any bubbles.
  8. Add 10ul of 5x LD to the PCR tubes of digest.
  9. Load the following to the wells.

 

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1

2

3

4

5

6

7

8

9

10

3 uL 1kb ladder

 

25 - 30 ul of digest

Add as needed

            

 

  1. Run gel (~160 volts) until purple has reached halfway down the gel.
  2. Examine gel under blue light in the bathroom. Take a picture! Make sure the band that has appeared is of the appropriate size
  3. Cut out desired bands. Cut off as much gel as you can from around the glowing band, but DO NOT CUT INTO BAND!
  4. Proceed immediately to the gel extraction protocol.

 

[Insert Picture of Gel]



Gel Extraction:

Materials:  

  • Qiagen Gel Extraction Kit [underneath bench 3 in box labelled “Qiagen Gel Extraction Kit iGEM”] Should contain:
    • Buffer QG
    • Buffer PE (with ethanol added)
    • Buffer EB
    • 2mL collection tubes
    • QIAquick spin columns
  • Gel slices digested _______[From last step]
  • Hot water bath [bench 4]
  • Centrifuge [bench 4]
  • Falcon tubes [storage closet]
  • Waste container [sink]
  • P1000 and P200 micropipettes [bench 3]
  • Scale [bench at the back of lab]

 

Procedure:

 

  • *Note: Preheat water bath to 50 C as soon as possible!!!

 

  • All centrifuging happens at 13,000 rpm

 

  1. Add 1000 ul of buffer QG to each tube.
  2. Incubate at 50 C in water bath until gel slices have completely dissolved. To help dissolve gel, mix by vortexing the tube every 2-3 minutes during the incubation.
  3. Place a QIAquick spin column in a provided 2 ml collection tube.
  4. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min at 13100 rcf.
  5. Discard flow-through and place QIAquick column back in the same collection tube.
    1. For samples larger than what will fit in the spin column, centrifuge and  repeat steps 6 and 7 until full sample has been applied and spun through column.
  6. Add 0.5 ml of buffer QG to QIAquick columns and centrifuge for 1 min.
  7. To wash, add 0.75 ml of buffer PE to column and centrifuge for 1 min.
  8. Discard the flow-through and centrifuge the column for an additional 1 min.
  9. Place column into a clean 1.5 ml microcentrifuge tube labelled appropriately with the plasmid and date.
  10. To elute DNA, add 30 ul of buffer EB to the center of the QIAquick column membrane and let it sit for 5 minutes. Centrifuge for 1 min.
  11. DNA should be present as a clear solution under the column.
  12. Discard column.
  13. Check concentration with NanoDrop.
  14. Record where tube is stored _______________, and what it’s labelled as: ________

Results:

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Label

Concentration

Volume

Location

Notes