Line 441: | Line 441: | ||
<b><h3> 4. Gel </b> </h3> | <b><h3> 4. 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 DNA runs to red. | + | <DT>4.1 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 DNA runs to red. |
− | Pour the TAE buffer evenly to completely cover the gel. | + | <DT>4.2 Pour the TAE buffer evenly to completely cover the gel. |
− | Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA to be put in each well. | + | <DT>4.3 Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA 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. | + | <DT>4.4 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. | + | <DT>4.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber. |
− | Ligation | + | <br> |
+ | |||
+ | <b><h3> 5. Ligation </b> </h3> | ||
+ | |||
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. |
Revision as of 04:21, 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