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 1˝ 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
The PfuTurbo® Cx Hotstart DNA polymerase is currently the only commercially available proofreading DNA polymerase which can amplify uracil containing templates.
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.
NB. Electrocompetent cells cannot be used as the electro shock will cause the hybridized DNA fragments to dissociate.
If you use this protocol please cite: Frandsen et al. 2008 BMC Molecular Biology
Dette sted blev sidst opdateret 14. July 2010