Re-suspend each pellet in 215 µl buffer P1 (cell resuspension solution).ģ.Ědd 215 µl buffer P2 (cell lysis solution) and invert the tube 8 times to mix.Ĥ.Ědd 330µl buffer P3 and invert 8 times to mix.Ħ.Ěpply 730 µl of supernatant to DNA column.ħ. Spin down overnight culture in microcentrifuge tubes and discard the supernatant.Ģ. (07/21/16) Plasmid Purification (Miniprep)ġ. Pick isolated colonies formed on the desA transformation plate and mix well with the media (pipette up and down). In culture tubes, take 4 ml of LB + spectinomycin media. (07/20/16) Prepare Bacterial Culture for Mini Prepġ.Ědd 50 ul of spectinomycin antiobiotic into 50 ml LBĢ. Result: No colony grew in SFR2 and tSFR2 plates, but there were several colonies grew in the desA plate. Spread plate 50 µl and 200 µl of them onto LB plates with spectinomycin antibiotic as the selective marker. Incubate in the 37☌ shaker for an hour.ĥ. Put it back on ice for another 2 minutes and add 900 µl of LB into the tube. Heat shock cells at 42☌ for 45 seconds.Ĥ. Add each tube of competent cells (50 µl) with 2.5 µl and 5 µl to 50 µl.ģ. Use 2 different volumes of Gibson product to mix with the competent cells.
Thaw competent cells on ice for around 5-10 minutes.Ģ. Set up the reaction in 50☌ for 30 minutes Transformation of the Recombinant Plasmid into DH5α Competent Cellsġ. We also run negative control where we just put the plasmid backbone without insert.Ĥ. Gibson Assembly master mix from NEB is used, so all we need to add is the plasmid backbone and the inserts according to the amount we calculated above in total reaction of 20 µl. We used 3 fold of excess insert, so the number of pmols for plasmid will be 3 times the amount of insert we need (0.036 pmols).ģ. Calculate the number of pmols needed for each fragment if we use 30 ng of the vectors. Plasmid Concentration: 8.1 ng/µl Gibson Assemblyġ. Gel extraction and concentration is measured with nano drop. Run Gel Electrophoresis with 0.8% agarose at 150 V for 30 minutes. Restriction digest for the riboswitch plasmid (3745 bp) with Fast Digest from NEB in total reaction of 20 ul. (07/19/16) Restriction Digest of Riboswitch Plasmid with EcoRI and XbaIġ. Then, DNA concentrations were measured with nano drop. Gel Extraction was done with Wizard SV Gel and PCR clean up kit by Promega.
SNAPGENE VIEWER RESTRICTION ENZYME DIGEST FULL
Run the PCR reaction with touch up program, where we run 7 cycles with the temperature of the primer without the overhang and another 30 cycles with the temperature of the primer full sequence.ģ. We also use 2 different buffers because we figured that our gene sequence has a high GC%.Ģ. Mix the following reagents for PCR amplification, each reaction of 50 µl and we did one negative control where we did not put the template DNA. The text area below.(07/18/16) PCR Amplification (SFR2, tSFR2, desA) with Phusion High Fidelity Polymeraseġ. Paste a raw sequence or one or more FASTA sequences into Restriction Map supports the entire IUPAC alphabet and Program as a reference when planning cloning strategies.
The translation of the DNA sequence is also given, in Map showing the positions of restriction endonuclease cut Restriction Map accepts a DNA sequence and returns a textual
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