TOPO?TA?Cloning

Instructions Modified for Simpson Lab

1.PCR:  Perform a standard PCR reaction using taq polymerase.  (Do not use tfl as the PCR product must have a 3' adenine overhang that only taq generates.)  Include an 72℃ extension step for 7 to 30 minutes at the end of the reaction to ensure that all products are full length and 3' adenylated.  Check results on a gel and proceed immediately to the cloning reaction.

2.Prepare for the Transformation Reaction.

a.Set water bath to 42℃.

b.Warm SOC medium (Box 2) to room temperature.

c.Warm pre-poured LB plates with 50 µg/ml ampicillin at 37℃ for 30 minutes.

d.Spread 40 µl of 40 mg/ml X-gal (in foil-coated box in -20℃ freezer) on each LB plate and incubate at 37℃ until ready for use.

e.Thaw 1 vial of One Shot cells for every three transformations.

3.Set up Cloning Reaction:  We do our reactions at 1/3 the manufacturer's recommended volumes, and so use one vial of One Shot cells for every three transformations.  The reaction mixture is as follows:

Fresh PCR product1.33 µl (from Step 1)

Salt Solution  0.33 µl (in kit in -20℃ freezer)

TOPO Vector 0.33 µl  (in kit in -20℃ freezer)

FINAL VOLUME2.00 µl

4.  Incubate:  Gently mix (do not use a pipettor!) the reactions and incubate at room temperature for 5 minutes.  For large PCR products (>1 kb) or for a pool of PCR products, you can increase the time up to 30 minutes.  After incubation, place the reaction on ice (or store overnight at -20℃).

5.Transform into Competent Cells:

a.Add 16 µl of the One Shot Chemically Competent E. coli cells to each cloning reaction.  Each vial of One Shot will yield enough for three cloning reactions.

b.Incubate on ice for 5 to 30 minutes.

c.Heat-shock the cells for 30 seconds at 42℃ without shaking.

d.Immediate transfer the tubes to ice.

e.Add 83 µl of the room-temperature SOC medium.

f.Incubate for 1 hour in the 37℃ shaking incubator at 200 rpm.

g.Using sterile technique, spread 40 µl from each transformation onto a prewarmed LB plate.  Let stand right-side up for about 5 minutes to allow the cells to adhere to the agar, then invert and incubate overnight at 37℃.

6.Select and Analyze Colonies:  Select and analyze only white or light blue colonies; dark blue colonies have not received inserts.  We analyze colonies via PCR.  There are two options for doing this which are listed below.  Sample 15-20 colonies and mark each with a number on the underside of the plate for reference purposes.

Option 1:  You can PCR directly from a colony.  Simply set up a standard PCR reaction, then take a plug from each colony to be sampled using a P10 pipet tip and a pipettor set to 10 µl.  You will have to pipet up and down once or twice to get the cells to detach from the pipet tip and into your PCR reaction.  This has the advantage of being quick and easy, but the colonies are frequently so messed up by the sampling that they are hard to resample should you need to do this.

Option 2:  Grow your colonies in microfuge tubes in 200 µl (or less?) LB broth with 50 µg/ml ampicillin at 37℃ in the shaking incubator for 1 or 2 days.  E. coli clumps can be dispersed by vortexing for a minute or two.  Cell suspensions should be cloudy when vortexed.  One µl serves as template for PCR reactions.  This takes more time than the first option, but has the advantage of allowing for multiple PCR reactions from the same colony.

Not all of the white colonies will have been transformed with full-length PCR product (they may have been transformed with primer or partial product), so not all will yield PCR product from Step 6.  (This is why you sampled more than 10 colonies.)  Of those that did amplify successfully, standard operating procedure is to sequence 10, although this can be varied depending on the purpose of the study and budgetary constraints.  (One option is to sequence one primer only for 10 colonies to assess variation, then to sequence the reverse primer only for one colony representing each unique sequence type.)