Based on James F. Smith protocols, Qiagen QIAQuick Gel Extraction protocol, and Qiagen PCR Cloning Handbook.

Preparation/Gel Extraction

Note: Ligation works best with fresh PCR products (<24 hours)

  1. Prepare one LB +Amp plate for each band (see Lab Recipes). Ampicillin is most potent and colony selection works best with fresh plates (<1 week).
  2. Run entire PCR product on a thick minigel (see Lab Recipes).
  3. Set heating block to 50C.
  4. Cut bands to be cloned:
    1. Label and weigh 1.7uL tubes before cutting bands so you can exactly calculate the weight of each band.
    2. Quickly(!) take a gel image (UV damages DNA and GelRed can’t be visualized on blue light). On the image, label the bands to be cut (a, b, c, etc… shortest to longest)—but just take notes now and edit the image later when the DNA isn’t being damaged by UV.
    3. Quickly(!) cut the gel on the 300nm UV box in AGRS266. Protect yourself with the thick plexiglass UV shield.
    4. Use a new blade for each band and avoid cross-contaminating samples.
  5. Add 3 volumes buffer QG to gel slice (1 uL for every mg of gel).
  6. Incubate at 50C for 15 minutes. Vortex the tube every 2-3 min to help dissolve gel.
  7. During the incubation, label a 1.7uL tube for each sample (for the final elution).
  8. Add 10uL 3M sodium acetate and mix.
  9. Add 1 gel volume isoproponol and mix.
  10. Place a Qiaquick spin column in a provided 2mL collection tube. To bind DNA, apply up to 800uL of the sample to the Qiaquick column and centrifuge for 1 min at max speed.
  11. Pour flow-through back into the column and spin through a second time for 1 min. Discard flow.through. Repeat for the rest of the sample if there is more than 800uL. If combining multiple bands of the same product, bind them all to the same column.
  12. To wash, add 750uL buffer PE to column. Let column stand 5 minutes. Centrifuge 1 min and discard flow-through.
  13. For a second wash, add 750uL buffer PE. Centrifuge 1 minute and discard flow-through.
  14. To remove residual wash buffer, centrifuge 1 minute. Rotate 180 degrees and spin a second time to remove the droplet that often adheres to column bottom. Discard flow-through with tube.
  15. Place Qiaquick column into a clean 1.7uL tube.
  16. To elute, add 30uL buffer EB to the center of the membrane and place in heatblock for 5-10 minutes. Centrifuge 1 minute.

Ligations

  1. Thaw on ice: 2X ligation master mix and pDrive cloning vector (-20 freezer).
  2. Label strip tubes and place on ice.
  3. Record tube contents on Ligation setup sheet.
  4. Pipette into each tube (in this order!):
    • 2uL ddH2O
    • 2uL PCR product
    • 1uL pDrive cloning vector
    • 5uL 2X Ligation master mix
  5. Mix solution by pipetting
  6. Close tubes and place in refrigerator for 30 minutes to 2 hours (more? longer=better).
  7. Proceed immediately to Transformations or store ligation products at -20.

Transformations (w/Qiagen EZ Competent Cells)

  1. Set heatblock to 42C and incubator to 37C.
  2. Thaw SOC medium, 100mM IPTG, and 40mg/mL X-gal (-20 freezer) to room temperature.
  3. Remove a LB +Amp plate for each transformation from the refrigerator. Label plates with the info from the Ligation setup sheet: PCR product & band (ex. 220-6b)
  4. Make an IPTG/X-gal master mix with the following quantities per plate:
    • 12.5uL IPTG solution (100mM)
    • 50uL X-gal solution (40mg/mL)
  5. Mix well and pipette 62.5uL of the master mix onto each plate. Spread over the entire surface with a flame sterilized glass spreader. Cover to protect from light and let sit at least 30 minutes before using plates.
  6. Label a 1.7mL tube for each reaction and place on ice.
  7. Thaw Qiagen EZ Competent Cells (-80 freezer) on ice. Treat them gently. NOTE: This protocol is specific to these cells.
  8. Spin down ligations.
  9. Into each tube place:
    • 25uL competent cells (pipette gently!)
    • 2uL ligation product (move the tip through the cells as you eject and then stir gently with the tip).
  10. Close caps and place on ice 5 minutes. Put remaining ligation product in the refrigerator.
  11. Heat shock by placing tubes into 42C heatblock for 30 seconds.
  12. Immediately transfer tubes back to ice for 2 minutes. Turn off heatblock.
  13. Add 150uL room temperature SOC medium to each tube.
  14. Pipette 125uL of transformation onto the plate and spread with sterilized (but cool) spreader.
  15. Place plates in 37C incubator, agar side down.
  16. After 1 hour flip plates upside-down and let them sit overnight. Place remaining transformations in refrigerator with the ligations.
  17. After plates incubate overnight, transfer to refrigerator for 1 hour (or more) to enhance blue color development before proceeding to colony PCR.

Colony PCR

  1. On ice, prepare master mix according to cloning PCR template. One 25uL reaction for each clone to be amplified (+ one negative control + one for pipette error).
  2. On the PCR sheet, identify samples with: PCR product and band (from the Ligation sheet and plate label), and a number for the clone (ex. 220-6b1, 220-6b2, 220-6b3…)
  3. Aliquot 25uL master mix into PCR tubes.
  4. Set a pipette to 8uL and touch the tip to one of the white colonies surrounded by blue colonies. Place the tip into the reaction tube and pipette up and down to release cells.
  5. Use “ta cloning” program on the thermocycler.
  6. Store plates in the refrigerator.
  7. Visualize on a standard minigel and clean up products with standard PCR purification kits.
  8. If band lengths are as expected, ligations and transformations can be discarded. Save plates in refrigerator until sequencing is complete.