14/07/10

Preparation of vectors for USER Friendly cloning  (please see 'media' for details)

 Production of pRF-HU2 and pRF-HU2E vectors:

The pRF-HU2 and pRF-HU2E vectors are maintained as low copy number plasmids in E. coli. The protocol requires 10 µg vector, why it is necessary to maxiprep the vectors from 3 L of LB culture.

             1.       Inoculate a 10 ml LB + kanamycin A (25 mg/ml) starter culture with a single colony and incubate overnight at 37 ^oC with 200 rpm stir.

 2.       Inoculate a 3 L of LB + kanamycin A (25 mg/ml) culture with 3 ml of overnight starter culture in the late afternoon and incubate overnight  at 37 ^oC with 200 rpm stir.

             3.       Harvest the culture with centrifugation at 6000 x g for 10 minutes at 4 ^oC.  Remove the supernatant.

             4.       Purify the plasmid using a suitable technique. We normally use the Qiagen ‘HiSpeed Plasmid Maxi Kit’ (Qiagen cat. no. 12662 or 12663), following the manufactures recommendations.

    Linearization of the vectors:

1.       Digest 10 µg pRF-HU2 or pRF-HU2E with 70 units (7 µl) PacI (New England Biolabs, Cat. no. R0547) overnight at 37 °C in a total volume of 300 µl with NEBuffer 4 + BSA.

2.       The next day, add an additional 20 units (2 µl) of PacI and 40 units (4 µl) Nt.BbvCI (New England Biolabs, Cat. no. R0632), and incubate for 1 hour at 37 °C.

3.       Verify the linearization of the vector by gel electrophoresis (2-3 µl). NB. When you include the time to run the gel electrophoresis the total digestion time with Nt.BbvCI will amount to 1½ hour which is more than sufficient.

4.       Purify the linearized vector using the ‘illustra GFX PCR DNA and Gel Band Purification kit’ from GE healthcare (Cat. no. 28-9034-70 or 28-9034-71). Use two mini spin columns to purify the 300 µl and elute the DNA in 50 µl of EB buffer.

5.       Determine the concentration by gel electrophoresis and measure it on a spectrophotometer

This protocol normally results in a DNA concentration of app. 50 mg/ml and a volume of 90 µl, sufficient for 20 cloning reactions. Divide the app. 90 µl into four aliquots and store them at -20 ^oC until use, this will prevent degradation of the DNA by repeated rounds of  freezing and thawing.  

USER Friendly cloning reaction                      

1. Perform PCR with PfuTurbo® C_x Hotstart DNA polymerase (Stratagene) using the manufacturer’s recommendations in a reaction volume of 15 µl per reaction.

The PfuTurbo® Cx Hotstart DNA polymerase is currently the only commercially available proofreading DNA polymerase which can amplify uracil containing templates.

2. Check the success of the PCR reaction by loading 5 µl of the reaction volume on an agarose gel. Note that it is not necessary to purify the PCR amplicon before the USER Friendly cloning reaction.

    3. Mix the following in a 0.2 ml PCR tube

PCR product 1                      10 µl              (150 ng)

PCR product 2                      10 µl              (150 ng)

Linearized vector                    4 µl              (200 ng)

USER enzyme mix                 1 µl

Total volume                        ~ 25 µl

It is not necessary to add any buffer to the reaction as the USER enzymes can function in most 1 x DNA polymerase buffers. However, if you are forced to purify the PCR products due to many unspecific bands you should add DNA polymerase buffer to the reaction.

4. Incubate at 37 °C for 20 min followed by 25 °C for 20 min (we use a PCR cycler for this).

5. Transformation:

1. Transfer the reaction mix to a pre-cooled 1.5 ml Eppendorf tube.
2. Add 50 µl of chemically competent E. coli JM109 cells (> 1*10⁶ cfu) to the Eppendorf tube.
3. Mix gently by tapping on the tube a couple of times and incubate for 30 minutes on ice.
4. Heat shock the cells by incubating them at 42 ^oC for 45 seconds, immediately afterwards return the cells to ice and incubate for 2 minutes.
5. Add 450 µl SOC medium, mix by inverting the tube a couple of times and incubate for 1 hour at 37 ^oC with 300 rpm. 
6. Pellet the cells in a table top centrifuge (1 minute at 5.000 rpm), discard 9/10 of the supernatant and resuspend the cells by pipetting up and down until all cells have been separated. Plate the approximately 50 µl onto a single LB plate supplemented with 25 µg/ml kanamycin A.
7. Incubate the plate overnight at 37 ^oC.

NB. Electrocompetent cells cannot be used as the electro shock will cause the hybridized DNA fragments to dissociate.

6. The next day: Isolate the obtained colonies onto a new LB plate supplemented with 25 µg/ml kanamycin A and incubate the plate overnight at 37 ^oC.
7. The next day: Screen 5-10 of the resulting colonies by PCR using the insert specific primers used to amplify the two inserts in step 1 (two reactions pr colony). It is also possible to perform the screening on the plates from step 6, however, we normally get a very high level of background due the free amplicon DNA found on the plates from the cloning reaction.
8. Chose 1-5 colonies that are positive for both inserts and setup 10 ml liquid LB cultures supplemented with 25 µg/ml kanamycin A and incubate the plate overnight at 37 ^oC.
9. Purify plasmids and check the size by restriction enzyme digestion
10. Sequence the insert (use RF-1 and RF-2 for pRF-HU2 and RF-3 and RF-2 for pRF-HU2E)

 If you use this protocol please cite: Frandsen et al. 2008 BMC Molecular Biology