Introduction to cloning

Transcription

1 1 of 14 Introduction to cloning Aim The aim of this protocol is to serve as a general guideline to mainstream molecular cloning of Gene of Interest ( GOI ). Overview GOI Sequence Transformation into Bacteria Choose Vector Primer design Seed transformants on selection media Amplification of GOI ("Insert") Pick selected colonies Colony PCR Restriction of Insert Restriction of vector Seed transformants on selection media Dephosphorylation of ends Grow Starter from positive clones PCR purification kit PCR purification kit Mini-prep of Plasmid DNA Sequencing Ligation

2 2 of 14 Experiment procedure Primer design 1. Choose appropriate vector: Type of expression drive (promoters) Type of Tags and their location in regard to Multiple Cloning Site (MCS) (N or C terminal) Appropriate restriction sites in MCS which do not exist in GOI 1 Direction of expression Existence of Ribosomal Binding Site (RBS) if needed 2. Choose restriction site(s): Preferable, choose 2 different restriction sites within the MCS (and do not restrict the GOI). Usually sticky ends are a better choice than blunt ends Check and see if these enzymes are in stock, and if not order or choose a different set. 3. Design the forward primer - using a text editor or BioEdit software 2 copy the 5 3 restriction site. 4. Copy approximately base pairs of the GOI s start sequence to the 3 end of the restriction site (verify you don t forget the Met codon sequence!) This design will create an insert construct that will contain the GOI with the needed restriction sites 5. Add between 3-6 repeats of base pairs at the 5 end of the restriction site; make sure the sequence maintains the correct reading frame. 1 If such a case is not possible, choose a restriction site that cuts the GOI at middle; after restriction, separate the cut insert on a gel and cut the pool of insert that were not restricted at the middle of the gene 2

3 3 of 14 If possible, add a strip of Guanine or Cytosine (or a mix); make sure the strip at end of the reverse primer will not complement, to ensure no primer-dimer formation Total length of primers should be from 24 to 40 base pairs; add or remove base pairs to change the physio-chemical properties of the primer and it s melting temperature (T m ) (Tm should be calculated only for complementary sequences without the restriction sites sequences). Try to aim at Tm between C. 6. Design the reverse primer repeat steps 3-5. Make sure the stop codon is included at the end of the reverse primer! (upstream to the restriction side). IF a tag is to be added to the C terminal, make sure to remove the stop codon from the primer sequence. 7. Reverse-complement the obtained sequence (use BioEdit or OligoCalc) 8. Test primers for properties via OligoCalc: Try to keep GC content above 40% and below 70% Check that the primers set do not form primer-dimer and secondary structure (check that the 3 ends of each primer do not complement the other primer). 9. Calculation of appropriate Ta: Average the T m of both primers Subtract 5 C to get the initial Ta; Via gradient option, test amplification at a range of (-10 C) to (+5 C) of the initial Ta (2-3 C intervals). 10. On oligo arrival, centrifuge vial at maximum speed Reconstitute oligo with sterile DDW aiming for 100pmol/µl concentration of each primer (master stock X500). Meaning, if the amount of the primer is 32nmol, reconstitute primers with 320µl of DDW. Dilute master stock 1:10

4 4 of 14 with DDW/MQ and divide into several aliquots of 100µl volume (common primers should be completely divided to smaller aliquots); store at -20 C. Amplification of GOI 1. Equilibrate the following ingredients to room temperature (except Taq enzyme): PCR recipe at 25µl volume Ingredient Volume (µl) Bufferx10 * 2.5 dntps (final concentration 0.2mM each) Primers (each) 0.5 (total 1µl) Sterile DDW X Template (min 50ng plasmid DNA/200ng of Y genomic DNA) Taq enzyme (5U/µl) 0.5µl *- Make sure Mg 2+ is included in the buffer See appendix C for extraction protocol of genomic DNA from bacteria. 2. It is recommended to add a negative control which contains no template (blank sample) and a positive control (a set of primers-template that is known to amplify at a similar thermal settings). 3. A general amplification setup will include the following conditions: a. 95 C -> 2' b. 95 C -> 30'' c. Gradient Ta - > 30'' d. 72 C -> 30'' (repeat step "b-d" cycles) e. 72 C -> 10' 3 Preparation of dntp s mix see appendix A

5 5 of 14 f C -> Hold Another option is to use touchdown PCR according to the following scheme 4 : 4. While the PCR is running, prepare Agarose gel according to the expected amplicon size (see appendix A for recipe). 5. Separate PCR products (load approximately 5µl) and evaluate amplicon size, homogeneity of products and amplification intensities; fine tune annealing temperature if there is no major correctly sized band detected. Restriction of GOI & Vector 1. Mix the following ingredients in eppendorf tubes (parallel restriction of both plasmid and insert): Endonuclease restriction recipe at 50µl volume Ingredient Volume (µl) PCR products/ Plasmid DNA (minimum 3µg) 20/X Bufferx10 * 5 BSAx10 (if needed!) 5? Restriction Endonuclease 1 DDW Fill to 50 4 Adopted from Tolia NH, Nature Methods Vol. 3, No. 1, 2006

6 6 of 14 *- Make sure to use the optimal buffer (100% activity; refer to product s leaflet)t It is possible to do double restriction with two restriction enzymes that share compatible buffer for 100% activity 2. Incubate at 37 C for 1-1.5hr 6. (optional): if there are several bands in the reaction, remove the band from the gel using UV-table set to 70% intensity (work quickly: UV light promotes DNA damage and mutagenesis). 3. Apply phosphatase (Calf intestine phosphatase, Cat# M0290S, NEB) to plasmid DNA by mixing 1µl of CIP in the reaction mix and incubating for additional 1hr at 37 C. 4. Mini-elute PCR product with Bioneer s PCR isolation kit (or similar kit from other manufacturers) according to manufacturer s protocol; Elution should be performed with sterile DDW at 22µl volume; incubate 10 at RT. 5. Measure sample concentration via Nanodrop (1-1.5µl sample). Ligation of GOI and Vector 1. Perform ligation at 20µl volume with 1:3 or 1:5 ratio (in favor of insert) when insert and vector are not similar in length (most cases): Ligation recipe at 20µl volume Ingredient Volume (µl) Insert (according to ratio) Y Plasmid DNA (50ng-100ng) X Bufferx10 2 T4 DNA ligase (20U/µl) 1 DDW Fill to 20

7 7 of 14 Ratios of insert/vector should be calculated as follows 5 : ng Vector Kb Insert α = ng Insert Kb Vector For example, a ratio of 5:1 in favor of insert (770bp) to vector (4800bp) : 100ng 0.77Kb 5 = 26.7ng 4.8Kb Incubate at 4 C O.N 6 Next day introduce ligation sample into DH5α or other appropriate competent bacteria (see protocol#2 for electroporation or printed heat shock protocol; see appendix D for quick preparation of electrocompetent cells) Seed 100µl of the inoculum on half of the plate and then spin rpm and seed 20µl on the other half of the plate Selection of candidate clones via colony PCR 1. Next day, examine plate and evaluate number of growing colonies. If there are numerous colonies, choose 5-10 isolated colonies and mark their location on the plate with index numbering. 2. Setup PCR reactions equal to the number of colonies with all ingredients including Taq and appropriate primers; place on ice. 3. Pick between 5-10 isolated and small colonies each pick, mixed several times in a tube and then spread on half a plate with appropriate antibiotics. 4. Place tubes in thermal cycler and plates for O.N incubation at 37 C. 5. Load PCR products on agarose gel and evaluate the amplicon size. 5 α is the [insert/vector] ratio 6 Also possible at 16 C (Takara); T4 Ligase of NEB use O.N at 4 C

8 8 of 14 Miniprep and Sequencing 1. Prepare starters from positive clones in 6ml LB + appropriate antibiotics. 2. Remove 750µl of inoculum and mix it with 250µl of 80% glycerol; vortex vigorously and flash freeze with liquid nitrogen. 3. Remain of inoculum will be used to isolate plasmid DNA via miniprep (Bioneer, see previously). 4. Established plasmid DNA should be sent to sequencing (minimum of 500ng) notify Geula in regard to the amount of samples (note that sequencing with two primers is regarded as two reactions).

9 9 of 14 Appendix A Quickchange Site-directed mutagenesis Aim In-vitro Site-directed mutagenesis (SDM) is a relatively easy and quick technique to introduce point mutations, insertions or deletions into a plasmid. In SDM, the pfu DNA polymerase is utilized to amplify with proofreading capabilities long stretches of double stranded DNA (such as whole plasmids). A set of long primers (>40bp) are used to amplify the plasmid while introducing the mutagenesis (whether it is a switch of base pairs or a deletion/insertion). After completion of the PCR amplification, the mixture of original plasmid and newly-mutagenized plasmid are incubated with DpnI restriction Endonuclease, which digests only methylated nucleotides (only available on bacterial-synthesized plasmids), thus leaving the mutagenized plasmid intact. At this point, simple transformation assay can be conducted into the bacteria of choice followed by downstream applications. Tips for successful SDM Design the forward primer which have at least 15 base pairs that flank the point mutation on each side in case the mutagenesis requires more than three base pair exchange, use at the least 25 base pairs on each side (keep the flanking sequences always longer than the mutation itself). Copy the reverse complement sequence of the forward primer. Check the melting temperature (Tm) of the flanking sequence in online programs such as OligoCalc 7 (refer to salt adjusted values) see that the temperature is between 78 C-88 C. Check that the primers do not form secondary structures and hetero and homodimers close to the native G energy levels (IDT s OligoAnalyzer 8 )

10 10 of 14 General procedure 1. Setup PCR reaction as follows: SDM at 50µl reaction volume Ingredients Volume DDW 36µl Pfu Bufferx10 5µl dntps 2µl F primer 2.5µl R Primer 2.5µl Template (min 100ng) 1µl Pfu (2.5 U/µl) 1µl 2. Thermal settings 9 : a. 95 C 30 b. 95 C 30 c. 55 C d. 72 C Y (back to b x25cycles) (1min/1kb of dsdna) e. 72 C Digest w.t. plasmid DNA with the addition of 2µl DpnI restriction enzyme; incubate at 37 C for 2hrs. 3. Perform mini elution and elute plasmid DNA with 10µl of DDW incubated at RT for Transform bacteria via Heat shock/electroporation methods. 9 These are long PCR reactions thus it is recommended to run the reaction O.N 10 If encountered with problems change the Tm according to the calculated Tm

11 11 of 14 Appendix B Buffers, solutions and misc Tris Borate EDTA buffer (TBE) x10 Buffer/salt Molecular weight (gr/ml) Amount (gr) Tris Boric Acid Na 2 EDTA (0.5M) ph=8 40ml DDW (L) 1L Agarose gel Recommended Agarose gel concentrations for the separation of Nucleic acids Fragment size Recommended % Mass of Agarose (50ml volume) bp 3%-1.5% 1.5gr-0.75gr 1000bp-5000bp 1.5%-0.5% 0.75gr-0.25gr 1. Weigh the required amount of Agarose and pour powder into 250ml flask Using volumetric cylinder measure 50ml of TBEx1 and pour into flask. 3. Swirl several times and place in microwave for 1 ; watch that the Agarose doesn t spill over. 4. Arrange casting box with appropriate comb. 5. Once the gel solution is reached boiling temperature, cast it in the casting box and let the gel solidify. Running gel: Remove comb and submerge in TBEx1 filled running chamber 11 For 50ml gel volume; Use ratio of 1:5 between gel volume and flask s volume

12 12 of 14 Markers loading volume: 8-10µl Recommended max voltage: 180V Ethidium Bromide incubation time: minimum 30 dntps master mix (Invitrogen, Catalog# ) 1. Thaw freezed tubes (datp, dctp, dgtp, dttp at 100mM concentration) 2. Remove 100µl of each vial into an eppendorf tube and complete to 1ml with DDW (10mM). 3. Aliquot 50µl into PCR tubes and keep at -20 C.

13 13 of 14 Appendix C Quick extraction of genomic DNA from Bacteria 1. Pick a chunk of glycerol stock bugs and streak a plate with appropriate antibiotics. 2. Place it 37 C. 3. Next day, pick a colony with a tip and dip it into 40µl of DDW pipette vigorously till bacteria are evenly dispersed. 4. Heat a dry block to 95 C and Boil for Centrifuge tube at maximum speed for 5 at RT. 6. Take between 3-5µl into PCR tube and perform PCR amplification.

14 14 of 14 Appendix D Quick preparation of electrocompetent cells 1. Pick freshly grown colony with a sterile tip and dip into 50µl of sterile LB; mix vigorously till bacteria is evenly dispersed. 2. Mix this volume with 10ml of LB in a 50ml falcon tube and shake at 37 C till medium is turbid (2-3hr) rpm for 4 C. 4. Wash pellet x3 with 1ml of sterile DDW (repeat centrifuge setup at step 3). 5. Resuspend in 60µl of sterile DDW and perform electroporation.