Line 422: | Line 422: | ||
<br><br> | <br><br> | ||
<b><h3> 2. Nanodrop </b> </h3> | <b><h3> 2. Nanodrop </b> </h3> | ||
− | <DT>2.1 Vortex before nanodrop. | + | <DT><span>2.1 Vortex before nanodrop.</span> |
− | <DT>2.2 Wipe down the nanodrop machine with Kimtech wipes to make it sterile. | + | <DT><span>2.2 Wipe down the nanodrop machine with Kimtech wipes to make it sterile.</span> |
− | <DT>2.3 Set the program to analyze nucleic acids [because you are dealing with plasmid DNA]. | + | <DT><span>2.3 Set the program to analyze nucleic acids [because you are dealing with plasmid DNA].</span> |
− | <DT>2.4 Do a blank test to ensure that the platform is sterile. | + | <DT><span>2.4 Do a blank test to ensure that the platform is sterile.</span> |
− | <DT>2.5 Load 1uL of the miniprepped DNA onto the platform. | + | <DT><span>2.5 Load 1uL of the miniprepped DNA onto the platform.</span> |
− | <DD>2.5.1 (Have steady hands. The sample needs to be in the center for best results.) | + | <DD><span>2.5.1 (Have steady hands. The sample needs to be in the center for best results.)</span> |
− | <DT>2.6 Click “measure” on the nanodrop for analysis. | + | <DT><span>2.6 Click “measure” on the nanodrop for analysis.</span> |
− | <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><span>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).</span> |
<br> | <br> | ||
<br> | <br> | ||
<b><h3> 3. Digest </b> </h3> | <b><h3> 3. Digest </b> </h3> | ||
− | <DT>3.1 Dilute up to 1ug DNA to 17uL with dH₂O. | + | <DT><span>3.1 Dilute up to 1ug DNA to 17uL with dH₂O.</span> |
<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>3.1.1 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 | <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. | + | <DT><span>3.2 Use a microcentrifuge tube to put the reaction in. Put in the contents in this order: water, DNA, enzymes.</span> |
<DD>3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme. | <DD>3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme. | ||
<DD> 3.2.1.1 E and X = 10X RE-Mix | <DD> 3.2.1.1 E and X = 10X RE-Mix | ||
<DD> 3.2.1.2 S and P = standard enzymes | <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. | + | <DT><span>3.3 Incubate at 37℃ for 1 hour for standard enzymes, then at 80℃ for deactivation.</span> |
<br> | <br> | ||
<br> | <br> | ||
<b><h3> 4. Gel </b> </h3> | <b><h3> 4. Gel </b> </h3> | ||
− | <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. | + | <DT><span>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.</span> |
− | <DT>4.2 Pour the TAE buffer evenly to completely cover the gel. | + | <DT><span>4.2 Pour the TAE buffer evenly to completely cover the gel.</span> |
− | <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. | + | <DT><span>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.</span> |
− | <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. | + | <DT><span>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.</span> |
− | <DT>4.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber. | + | <DT><span>4.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.</span> |
<br> | <br> | ||
<br> | <br> | ||
<b><h3> 5. Ligation </b> </h3> | <b><h3> 5. Ligation </b> </h3> | ||
− | <DT>5.1 Use Antartic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure. | + | <DT><span>5.1 Use Antartic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure.</span> |
− | 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.</span> |
− | <DT>5.2 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. | + | <DT><span>5.2 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.</span> |
− | <DT>5.3 The ligase buffer should be thawed and resuspended at room temperature. | + | <DT><span>5.3 The ligase buffer should be thawed and resuspended at room temperature.</span> |
<DD>5.3.1 Gently mix by pipetting up and down and microfuge briefly. | <DD>5.3.1 Gently mix by pipetting up and down and microfuge briefly. | ||
− | <DT>5.4 Incubate at room temperature for 1 hour at 37℃ | + | <DT><span>5.4 Incubate at room temperature for 1 hour at 37℃</span> |
<br><br> | <br><br> | ||
<b><h3> 6. Transformation, Plate </b> </h3> | <b><h3> 6. Transformation, Plate </b> </h3> | ||
− | <DT>6.1 Thaw materials on ice for 5 minutes. | + | <DT><span>6.1 Thaw materials on ice for 5 minutes.</span> |
− | <DT>6.2 Put 10uL of ligation mixture into 100uL competent cells in a microcentrifuge tube. | + | <DT><span>6.2 Put 10uL of ligation mixture into 100uL competent cells in a microcentrifuge tube.</span> |
− | <DT>6.3 Flick the tube to mix. | + | <DT><span>6.3 Flick the tube to mix.</span> |
− | <DT>6.4 Put on ice for 30 minutes. | + | <DT><span>6.4 Put on ice for 30 minutes.</span> |
− | <DT>6.5 Add 200uL of LB media. | + | <DT><span>6.5 Add 200uL of LB media.</span> |
− | <DT>6.6 Incubate at 37℃ for one hour. | + | <DT><span>6.6 Incubate at 37℃ for one hour.</span> |
− | <DT>6.7 Plate 150uL of cells onto a plate. Make sure plate has the correct antibiotic (based on vector backbone)! | + | <DT><span>6.7 Plate 150uL of cells onto a plate. Make sure plate has the correct antibiotic (based on vector backbone)! |
− | Grow overnight. | + | Grow overnight.</span> |
<br><br> | <br><br> | ||
<b><h3> 7. Colony PCR </b> </h3> | <b><h3> 7. Colony PCR </b> </h3> |
Revision as of 15:46, 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