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<DT>2.6 Click “measure” on the nanodrop for analysis. | <DT>2.6 Click “measure” on the nanodrop for analysis. | ||
<DT>2.7 Write down measurements for the concentration of DNA (in ng/uL), A260, A280, 260/280 (should be around 1.8), and 260/230 (should be around 2.1). | <DT>2.7 Write down measurements for the concentration of DNA (in ng/uL), A260, A280, 260/280 (should be around 1.8), and 260/230 (should be around 2.1). | ||
+ | <br> | ||
+ | |||
+ | <b><h3> 3. Digest </b> </h3> | ||
+ | |||
+ | <DT>3.1 Dilute up to 1ug DNA to 17uL with dH₂O. | ||
+ | <DD>3.1.1 Take concentration of DNA from nanodrop and convert from ng/uL to ug/uL. Next, set up a proportion to find out how many uL you need to get 1 ug of DNA. | ||
+ | <DD>3.1.2 20uL (total reaction) - 2uL RE-Mix - 1uL standard enzyme = uL dH₂O | ||
+ | <DT>3.2 Use a microcentrifuge tube to put the reaction in. Put in the contents in this order: water, DNA, enzymes. | ||
+ | <DD>3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme. | ||
+ | <DT><DD>3.2.1.1 E and X = 10X RE-Mix | ||
+ | <DT><DD>3.2.1.2 S and P = standard enzymes | ||
+ | <DT>3.3 Incubate at 37℃ for 1 hour for standard enzymes, then at 80℃ for deactivation. | ||
+ | <br> | ||
+ | |||
+ | <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. | ||
+ | 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. | ||
+ | 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. | ||
+ | Ligation | ||
+ | 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. | ||
Revision as of 04:18, 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