Creating pentr vectors by BP reaction
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1 Creating pentr vectors by BP reaction Tanya Lepikhova and Rafael Martinez Overview: 1. Design primers to add the attb sites to gene of interest 2. Perform PCR with a high fidelity DNA polymerase 3. Purify PCR product 4. Perform a BP reaction of the gene into pdonr Transform the BP reaction into DH5-alpha 6. Verify contructs Example: In the following example we are going to generate a pentr vector inserting a gene of 2500 pb by BP reaction into pdonr221 kanamycin resistant (Invitrogen ). 1. Design primers to add the attb sites to gene of interest Take in count that if you want to express your gene in Eukaryotic systems a Kozak consensus sequence is needed. If you want to express the gene in Procaryotic systems add a Shine-Dalgarno consensus sequence. If wished both sequences can be added to the primers. The primers for a BP reaction must contain: 1. Four guanine (G) residues at the 5 end followed by 2. The 25 bp attb1 site followed by 3. At least bp of template- or gene-specific sequences C-terminal fusion proteins. If you want to express your protein with a C-terminal fusion tag do not add a stop codon. N-terminal fusion proteins. To express protein with N-teminal tag be sure that the two codons for Lys (AAA AAA) are in frame with start codon (see next page). This primer will work also for C-terminal fusion proteins. For more details check the Invitrogen Manual. Some examples are provided in the following page:
2 Primer design examples for BP reaction
3 2. Perform PCR with a high fidelity DNA polymerase Use a high fidelity enzyme to generate a PCR product i.e Phusion (Finnzymes F-530S) or TaKaRa Ex Taq (TaKaRa RR006A). Perform 2-4 reactions with a reaction volume of 50µl each. 3. Purify PCR product Check in agarose gel that the PCR product is of the expected size. If everything is correct proceed to clean the rest of the PCR product with a spin column (i.e Qiagen). Continue with the BP reaction. Note: if the BP reaction does not work repeat the PCR reaction and clean the product by cutting the band from an agarose gel. 4. Perform a BP reaction of the gene into pdonr221 To reduce background in the BP reaction linearize the pdonr with EcoRI. This enzyme cuts right in the middle of the gateway cassette. Inactivate the enzyme incubating at 65 C. Calculate 50 fmol and 100 fmol of your PCR product by using the following formula: where N is the size of the DNA in bp. Some examples are shown in the following table: Note: 50fmol of pdonr221 are 150ng
4 BP reaction Material: Gene of interest with att sites added by PCR Linearized pdonr221 (Invitrogen ) BP clonase (Invitrogen ) TE 1x Buffer ph 8.0 Proteinase K (included in the BP clonase kit) Positive control pexp7-let (included in the BP clonase kit) BP Reaction: Perform the BP reaction testing 50 and 100fmol of PCR product and pdonr221. Also include pexp7- let as a positive control. A typical reaction is as the following: 5. Transform the BP reaction into DH5-alpha Material: Method: Library Efficiency DH5α (Invitrogen ) BP reaction SOC medium without antibiotics LB agar plates with + Kanamycin 50µg/ml L-Spreaders Thermal Block at 42ºC. 1. Thaw the bacteria on ice (10-15min). Keep the bacteria on ice at any time!. Aliquot 20µl for each reaction in 1.5 ml eppendorf tubes. If there is still some bacteria left in the vial bring it back as soon as possible to -80 C.
5 2. Add 2µl from the BP reaction to 20µl competent cells and stir gently with the tip (do not pipette up and down!). Incubate 30min on ice. 3. Heat shock at 42 C for 30seconds. 4. Incubate on ice 3min. 5. Add 250µl SOC medium to the transformed competent cells and incubate at 37 C with shaking at 230rpm for 1hr. 6. While incubating label the LB-agar plates on the agar-side with: Gene/pENTR; Type of cell; Name and Date. 7. Spread 150µl of the transformed bacteria on agar plates with Kanamycin 50µg/ml. With a "L" tool spread uniformly all over the agar. Let the plate stand 5 min with the agar-side down to let the liquid be absorb. 8. Incubate overnight at 37 C with agar-side up. If everything is perfect colonies of around 0.9 mm will appear all over the plate. After incubation, plates can be store at 4 C for 1 month. TIP. Keep the tube from step 5 at 4ºC. In case there are too many colonies spread 50µl next day. TIP. Spreading 150 µl of bacteria in step 7 is a general rule. If there are difficult clones then spread the whole 250µl if necessary. 6. Verifying constructs 1. Pick up single colonies and incubate them in 5 ml LB medium with kanamycin 50µg/ml. Incubate overnight at 37 C with shaking at 230rpm. 2. Perform minipreps with 2 ml, store the rest of culture at 4ºC. 3. Verify your insert with enzyme digestion. 4. If everything is ok then prepare a glycerol stock (200µl Glycerol + 800µl media) and store at -80ºC. Digestion analysis with PstI and EcoRI of a pentr221 construct created by BP reaction of a PCR product of 2500pb into pdonr221. According with the expected digestion pattern samples on wells 3 and 4 have the right plasmid.
6 References: Cloning/GatewayC-Misc/Protocols.html
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