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Use Antartic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure. | Use Antartic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure. | ||
Make calculations using a 3:1 molar ratio of insert to backbone. Refer to the two tables below. | Make calculations using a 3:1 molar ratio of insert to backbone. Refer to the two tables below. | ||
+ | Put in each component in a microcentrifuge tube while on ice. They should be pipetted into the tube in this order: water, DNA, ligase buffer, ligase. | ||
+ | The ligase buffer should be thawed and resuspended at room temperature. | ||
+ | Gently mix by pipetting up and down and microfuge briefly. | ||
+ | Incubate at room temperature for 1 hour at 37℃ | ||
+ | <br> | ||
+ | <b><h3> 6. Transformation, Plate </b> </h3> | ||
+ | |||
+ | Thaw materials on ice for 5 minutes. | ||
+ | Put 10uL of ligation mixture into 100uL competent cells in a microcentrifuge tube. | ||
+ | Flick the tube to mix. | ||
+ | Put on ice for 30 minutes. | ||
+ | Add 200uL of LB media. | ||
+ | Incubate at 37℃ for one hour. | ||
+ | Plate 150uL of cells onto a plate. Make sure plate has the correct antibiotic (based on vector backbone)! | ||
+ | Grow overnight. | ||
+ | <br> | ||
+ | <b><h3> 7. Colony PCR </b> </h3> | ||
+ | |||
+ | Pick colonies with a combination of phenotypes i.e. large/small, red/white. Dilute each colony in 40uL dH₂O, 1uL DNA from ligation if transformation is successful. | ||
+ | If necessary, do a quick spin to make sure all the liquid is at the bottom. | ||
+ | Make the following master mix on ice in this order: 63uL dH₂O, 20uL buffer, 5uL VF₂ primer, 5uL VR primer, 2uL dNTP, 1uL Q5 polymerase. | ||
+ | Aliquot the master mixes into PCR tubes, then add 1uL of the DNA dilution. | ||
+ | Make sure PCR tubes are labeled properly and carefully! | ||
+ | Transfer the PCR tubes to a PCR machine and begin thermocycling. | ||
+ | Initial Denaturation: 98℃ for 30 seconds | ||
+ | 25-35 Cycles: 98℃ for 5-10 seconds, 50-72℃ for 10-30 seconds, 72℃ for 20-30 seconds/kb | ||
+ | Final Extension: 72℃ for 2 minutes | ||
+ | Hold 4-10℃ | ||
+ | <br> | ||
+ | <b><h3> 8. Gel </b> </h3> | ||
+ | |||
+ | Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the PCR samples run to red. | ||
+ | Pour the TAE buffer evenly to completely cover the gel. | ||
+ | Using a micropipette, put 3uL of PCR samples in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more PCR sample to be put in each well. | ||
+ | Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes. | ||
+ | Turn on the power supply and make sure bubbles are rising on the sides of the chamber. | ||
+ | <br> | ||
+ | <b><h3> 8. Inoculate correct colony to liquid culture </b> </h3> | ||
+ | |||
+ | Get the remaining 39uL of colony dilution. | ||
+ | Get LB media and make sure to use the appropriate antibiotic resistance. | ||
+ | Mix the colony dilution into the media. | ||
+ | Grow overnight. | ||
Revision as of 04:34, 19 October 2016
Experiments
Workflow
1. Miniprep/Nanodrop
2. Digest
3. Gel
4. Ligation
5. Transformation, Plate
6. Colony PCR (Screening)
7. Gel
8. Inoculate correct colony to a liquid culture.
Materials:
Miniprep: grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips
Nanodrop: nanodrop machine, miniprepped DNA, Kimtech wipes, micropipette and tips
Digest: miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA
Ligation: vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips
Transformation: ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips Plate: agar plate, micropipette and tips, beads
Colony PCR: dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA
Inoculate: LB media, dilution, micropipette and tips