Dahlquist:TOPO TA Cloning

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Protocol for TOPO TA Cloning.

Prepare Media

  • For pUC19 transformation control, need LB amp100 plates.
  • For control vector-only and vector+PCR, need LB kan50 plates.
  • For experimental samples, need LB amp50 plates or LB kan50 plates.
  • Conclusion: make LB kan50 plates, but leave a couple plates without antibiotic so we can spread ampicillin on them later for the pUC19 transformation.
  • Also need liquid LB media.

LB plates

  • 1.0% Tryptone
  • 0.5% Yeast Extract
  • 1.0 % NaCl
  • pH 7.0

For 500 mL (makes ~24 plates) 

5 g tryptone
2.5 g yeast extract
5 g NaCl
  1. Dissolve in 450 mL MilliQ water with stir bar in beaker.
  2. Adjust pH of the solution to 7.0 with NaOH.
    • Add ~2 drops 10 M NaOH solution.
  3. Bring volume to 500 mL.
  4. Add 7.5 g agar to 2 L flask, add liquid media.
  5. Autoclave on liquid30 cycle (30 minutes) with water in tray.
  6. After autoclaving, cool to ~55°C in water bath.
    • Pour two plates without antibiotic for the pUC19 control (ampicillin will be spread onto the plate).
  7. Add 500 μL of 50 mg/mL kanamycin antibiotic (100X stock). Swirl gently to mix.
  8. Pour plates and stack. Let cure on the bench overnight.

LB media

  • 1.0% Tryptone
  • 0.5% Yeast Extract
  • 1.0 % NaCl
  • pH 7.0

For 500 mL (Aliquot 100 mL to 5, 125 mL bottles) 

5 g tryptone
2.5 g yeast extract
5 g NaCl
  1. Dissolve in 450 mL MilliQ water with stir bar in beaker.
  2. Adjust pH of the solution to 7.0 with NaOH.
    • Add ~2 drops 10 M NaOH solution.
  3. Bring volume to 500 mL.
  4. Aliquot 100 mL to 5, 125 mL bottles.
  5. Autoclave on liquid30 cycle (30 minutes) with water in tray.

Control PCR

For each control PCR cloning/transformation reaction, prepare two LB plates containing 50 μg/mL kanamycin. Do not use plates containing ampicillin because the control template is a plasmid that encodes ampicillin resistance and the template will be carried over to the cloning/transformation reactions.

Producing the control PCR product

  • To produce the 750 bp control PCR product, set up the following 50 μL PCR:
    • Two reactions, negative control with water as template, and the positive control with the provided template.
      • To one 200 μL PCR tube, add 1 μL of sterile MilliQ water as the template.
      • To the other 200 μL PCR tube, add 1 μL of control DNA template.
    • Make the Master Mix:
                                        X1         X2.5
template                               (1.0 μL)    ---- 
10X PCR Buffer                          5.0 μL     12.5  μL
dNTP Mix                                0.5 μL      1.25 μL
Control PCR Primers (0.1 μg/μL each)    1.0 μL      2.5  μL
Sterile MilliQ water                   42.0 μL    105.0  μL 
Apex Taq Polymerase (5 units/μL)        0.5 μL      1.0  μL
Total Volume                           50.0  μL
  • Add 49 μL of master mix to each PCR tube.
  • Amplify using the following cycling parameters (Program called "TOPO"):
    • Turn on the thermocycler at switch in back.
    • Put tubes in thermocycler, making sure that they are sealed well.
    • Close lid and push handle to the down position (you don't need to turn the knobs).
    • Select "RUN" (Push "Enter" button when "RUN" is highlighted on screen).
    • Push the down arrow button until the "TOPO" program is highlighted.
    • Push the "Enter" button.
    • Push the down arrow button until "RUN" is highlighted.
    • Push the "Enter" button.
    • Push the "Screen" button to the left of the screen to view the progress of the run.
  • Initial denaturation for 2 minutes at 94°C for 1 cycle
  • Denaturation for 1 minute at 94°C, then Annealing for 1 minute at 55°C, then Extension for 1 minute at 72°C; repeat all these steps for 25 cycles
  • Final extension for 7 minutes at 72°C for 1 cycle

Remove 10 μL from the reaction and analyze by 0.8% agarose gel electrophoresis in TAE Buffer with ethidium bromide. A discrete 750 bp band should be visible. Proceed to the Control TOPO® Cloning reactions.

TOPO Cloning

Set up 6 μL TOPO Cloning reactions as described below. 

                    Vector Only       Vector + Control PCR Insert     Vector + PCR Insert (for what you want to clone)
PCR Product             -                    1 μL                            4 μL (can use 0.5 -4 μL, make up the difference with water)
Water                   4 μL                 3 μL                            -
Salt Solution           1 μL                 1 μL                            1 μL
TOPO Vector             1 μL                 1 μL                            1 μL
Total Volume            6 μL                 6 μL                            6 μL
  1. Set up control TOPO Cloning reactions at room temperature as shown above.
    • Add the reagents in the order of water, salt solution, TOPO vector, and PCR product.
  2. Incubate the reactions at room temperature (22-23°C) for 5-30 minutes and place on ice (we used 30 minutes). You may store the TOPO Cloning reaction at -20°C overnight.
  3. Proceed to the transformation protocol below.

Transformation

Preparing for Transformation

For each transformation, you will need one vial of competent cells and two selective plates.

  • Equilibrate a water bath to 42°C for chemical transformation.
  • Warm the vial of S.O.C. medium from Box 2 to room temperature.
  • Spread 22 μL of 100 mg/mL ampicillin on two plain LB plates; pre-warm in 37°C incubator for at least 30 minutes. These are for the pUC19 positive transformation control, which is not kanamycin resistant.
  • Spread 40 μL of Fermentas Ready-to-Use X-Gal (20 mg/mL) onto 2 LB kan50 plates per transformation reaction; pre-warm in 37°C incubator for at least 30 minutes.
  • Thaw vials of One Shot Top10 competent cells on ice, one for each transformation reaction.

One Shot Chemical Transformation Protocol

  • Add the appropriate sample to separate vials of One Shot Top10 competent cells.
    • 2 μL of water for the no vector control.
    • 1 μL of pUC19 (10 pg/μL) for the positive transformation control.
    • 2 μL of the TOPO cloning reactions. Store the remainder of the reaction at -20°C.
  • Mix gently. Do not mix by pipetting up and down.
  • Incubate on ice for 5 minutes (or up to 30 minutes; we did 5 min.).
  • Heat-shock the cells for 30 seconds in a 42°C water bath without shaking.
  • Immediately transfer the tubes to ice.
  • Add 250 μL of room temperature S.O.C. medium.
  • Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour.
  • Plate the transformation mix onto plates.
    • Use two plates per transformation, spreading a low and high volume onto each to ensure that at least one plate has well-spaced colonies.
    • To 10 μL of transformation mix, add 20 μL of S.O.C. medium to allow even spreading.
    • For the second plate use 20 μL of the transformation mix.
    • Store the remainder of the transformation at 4°C.
    • Transformations are spread onto the following types of plates:
      • no vector control onto LB kan50.
      • pUC19 positive control onto LB amp100
      • vector-only, vector + control PCR, and vector + target PCR onto LB kan50 and X-Gal.
        • Note that the vector + control PCR should only be plated on LB kan50 plates because the template for the control PCR is an ampicillin-resistant plasmid which carries over into the transformation, making ampicillin-resistant colonies that don't have the right plasmid.
  • Incubate overnight at 37°C.
  • An efficient TOPO Cloning reaction should produce several hundred colonies. Pick ~10 white or light blue colonies for analysis. Do not pick dark blue colonies.
    • Chemically competent cells plated at 10 μL + 20 μL S.O.C. should have a transformation efficiency of ~1 x 10^9 cfu/μg DNA

Analyze Transformants

  1. Take 5 white or light blue colonies of each target reaction and streak them out onto LB plates with 50 μg/mL kanamycin and X-Gal. Incubate overnight at 37°C. Store plates at 4°C.
  2. Pick a single white or light blue colony from each of the streak plates and inoculate into 5 mL overnight cultures of LB medium + 50 μg/mL kanamycin.
    • Use sterile technique to add 5 mL of LB medium to a sterile culture tube.
    • Add 5 μL of 50 mg/mL (1000X stock) to the LB in each tube.
    • Inoculate with a single colony.
    • Grow overnight in a 37°C incubator, 250 rpm.
  3. Isolate plasmid DNA using Zyppy Plasmid Miniprep Kit.
  4. Run a 0.8% agarose/1X TAE/EtBr gel to confirm plasmid purification.
  5. Analyze the plasmids by restriction analysis to confirm the presence and correct orientation of the insert. Use a restriction enzyme or a combination of enzymes that cut once in the vector and once in the insert. (Run gel.)
  6. Run PCR with the plasmid as template using the Lac-13910-for-EM and Lac-13910-rev-EM primers to confirm 448 bp product. (Run gel).
  7. Submit plasmids that have been confirmed by both PCR and restriction analysis for sequencing.

Zyppy Plasmid Miniprep Kit Protocol

Buffer Preparation

  • Add 24 mL 100% ethanol (26 mL 95% ethanol) to the 6 mL Zyppy Wash Buffer concentrate, 48 mL 100% ethanol (52 mL 95% ethanol) to the 12 mL Zyppy Wash Buffer concentrate, or 192 mL 100% ethanol (208 mL 95% ethanol) to the 48 mL Zyppy Wash Buffer concentrate.
  • Neutralization buffer should be stored at 4-8°C.
  • The 7X Lysis Buffer may have precipitated. If this occurs, dissolve the precipitate by incubating the bottle at 30-37°C for 10-20 minutes and mix by inversion. Do not microwave!
  • If processing large numbers of samples, perform the lysis and neutralization steps in groups of less than or equal to 10 preps to avoid excessive lysis, which can result in denatured plasmid DNA.

Plasmid Miniprep

The following procedure should be performed at room temperature (15-30°C)

  1. Add 600 μL of bacterial culture grown in LB medium to a 1.5 mL microcentrifuge tube.
    • The Zyppy Plasmid Miniprep Kit may also be used with the classic centrifuge-based procedure for processing up to 3 mL of bacterial culture. The procedure should be modified as follows:
      • Centrifuge 1.5 mL of bacterial culture for 30 seconds at maximum speed.
      • Discard the supernatant.
      • Repeat the previous two steps as needed.
      • Add 600 μL of TE or water to the bacterial cell pellet and resuspend completely.
  2. Add 100 μL of 7X Lysis Buffer (blue), mix by inverting the tube 4-6 times, and incubate for 1-2 minutes. Do not vortex!
    • After addition of 7X Lysis Buffer, the solution should change from opaque to clear blue, indicating complete lysis.
  3. Add 350 μL of cold Neutralization Buffer (yellow) and mix thoroughly. Do not vortex!
    • Neutralization is complete when the sample is yellow throughout and precipitate has formed.
  4. Centrifuge for 4 minutes at greater than or equal to 11,000 x g.
  5. Place a Zymo-Spin IIN column in a Collection Tube and transfer the supernatant from step 4 into the Zymo-Spin IIN column.
    • Avoid disturbing the pellet containing cell debris.
  6. Centrifuge the Zymo-Spin IIN for 15 seconds at greater than or equal to 11,000 x g.
  7. Discard the flow-through and return the Zymo-Spin IIN column to the same Collection Tube.
    • Ensure the flow-through does not touch the bottom of the column.
  8. Add 200 μL of Endo-Wash Buffer to the column and centrifuge for 30 seconds at greater than or equal to 11,000 x g.
    • Discard the flow-through and return the Zymo-Spin IIN column to the same Collection Tube.
  9. Add 400 μL of Zyppy-Wash Buffer to the column and centrifuge for 1 minute at greater than or equal to 11,000 x g.
    • Discard the flow-through and return the Zymo-Spin IIN column to the same Collection Tube.
  10. Spin again for 1 minute at greater than or equal to 11,000 x g to get any flow through off of the column.
  11. Carefully transfer the column into a clean 1.5 mL microcentrifuge tube with the cap cut off, making sure that none of the flow-through is touching the column.
  12. Add greater than or equal to 30 μL of Zyppy Elution Buffer directly to the column matrix and incubate for one minute at room temperature.
  13. Centrifuge for 30 seconds at greater than or equal to 11,000 x g to elute the plasmid of DNA.
  14. Discard the column and transfer eluate to a 1.5 mL microcentrifuge tube with a lid. Place on ice for immediate use or store at -20°C.

Restriction Analysis of Recombinant Plasmids

  • pCR2.1-TOPO plasmid information on Addgene
  • To sequence clones, need 15 to 20 μL of purified plasmid and PCR DNA at concentrations between 50-100 ng/μL for Laragen
  • Restriction digests. Goal is to Cut once in vector and once in the insert to determine orientation of insert relative to the vector.
    • Single enzyme to accomplish this:
      • SpeI (USB) cuts once in the vector (after 262) and once in the insert (after 303/307). We have the enzyme USB Buffer M.
      • BtgI cuts once in the vector (after 1719) and once in the insert. Don't have enzyme.
      • NcoI cuts once in the vector (after 1719) and once in the insert. Don't have enzyme.
    • Cuts insert only and we have the enzyme
      • HhaI (FastDigest)
      • StuI (USB Buffer M)
    • Cuts vector-only and we have the enzyme
      • ApaI, 357-356 (3931 bp), polylinker
      • BamHI, 253-252 (3931 bp), polylinker
      • BssHII, 1845-1844 (3931 bp), not in polylinker (USB Buffer M)
      • EcoRV, 314-313 (3931 bp), polylinker
      • HindIII, 235-234 (3931 bp), polylinker
      • KpnI, 245-244 (3931 bp), polylinker
      • NotI, 329-328 (3931 bp), polylinker
      • ScaI, 2438-2437 (3931 bp), not in polylinker (USB Buffer H)
      • XbaI, 347-346 (3931 bp), polylinker
    • Viable combinations of restriction enzymes to generate 2 fragments, cutting once in vector and once in insert:
      • StuI generates 311 + 137 bp fragments out of 448 bp insert (USB Buffer M)
      • ScaI cuts at 2437 in vector
      • BssHI cuts at 1844 in 4379 vector
      • Look at buffer compatibility to choose
    • Cuts vector three times (thought it didn't cut vector)
      • DraI (USB Buffer M); Cuts after 2340, 3032, 3051, the 19 bp fragment between 3051 and 3032 will not be detectable, so would look like 2 fragments produced.
      • DraI generates 169 + 279 bp fragments out of 448 bp insert
    • EcoRI cuts on either side of TOPO insert site (USB Buffer H)
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