Difference between revisions of "Team:IIT Delhi/protocol"

1. Inoculate 5 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking at 200 rpm.
2. Pour 2 ml of the culture into a microfuge tube. Centrifuge at maximum speed(13400 rpm) for 4-5 minutes in a microfuge. Store the unused portion(if any) of the original culture at 4°C.
3. Remove the medium by aspiration leaving the bacterial pellet as dry as possible.
4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.
5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents well by inverting the tube.

   NOTE:

   1. Do not vortex!
   2. Do not let the reaction proceed for more than 1 minutes.
6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube in ice for 3-5 minutes(optional).
7. Centrifuge the bacterial lysate for 10 minutes at maximum speed in a microfuge. Collect the supernatant to a fresh 1.5 ml mctube.
8. (Optional) Add equal volume of phenol: chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes in a microfuge. Transfer the aqueous upper layer to a fresh tube.
9. Precipitate nucleic acids from the supernatant. Now add 2 volumes of ethanol at room temperature to the supernatant in a 1.5 ml MCT. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature(optional). Discard the pellet in the original 2 ml MCT.
10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 10 minutes in a microfuge.
11. Discard the supernatant by aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kim wipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube. Stand the tube for 10 minutes.
12. (Optional)Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at in a microfuge.
13. (Optional)Remove all of the supernatant by aspiration. Take care with this step, as the pellet sometimes does not adhere tightly to the tube.
14. (Optional)Remove any beads of ethanol from the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).
15. Store the tube for 10 minutes at 37 degrees without shaking.
16. Dissolve the nucleic acids in 20 μl of mQ ( 1/100 times the initial volume in the MCT) or TE (pH 8.0) [containing 20 μg/ml Dnas e-free RNase A (pancreatic RNase)] . Vortex the solution gently for a few seconds and store the DNA at -20°C.

1. Transfer the following solutions in a micro centrifuge tube.
2. Incubate the mixture at 37 o C for 1 h to overnight. Keep the tubes in -4o C freezer or in -20o C freezer, after the incubation.

   NOTE:

   1. 10X buffer should be added in the reaction such that its final concentration is 1x (e.g. - in a 50 ul reaction 5 ul of buffer would be added).
   2. Amount of DNA to be added depends on concentration and the amount of DNA to be digested.
   3. Make sure that the restriction enzyme does not exceed more than 10% of the total reaction volume, Otherwise the glycerol and the EDTA in the enzyme storage buffer may inhibit digestion process.
   4. Enzymes on ice, when not in freezer
   5. Mix by pipetting
   6. A 50 μl reaction volume is recommended for digestion of 1 μg of substrate
   7. Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability
   8. If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.
   9. Cleanup of the PCR fragment prior to restriction digestion is recommended. PCR components can inhibit enzyme activity. In addition, the polymerase present in the PCR is active during the digestion step, and can modify the newly created ends by blunting them.
   10. Some enzymes may bind tightly to the substrate DNA. This binding can result in smearing or the presence of unexpectedly high molecular weight bands on a gel. To prevent this, add SDS to a final concentration of approximately 0.1%, or use Gel Loading Dye, Purple (6x), which contains sufficient SDS to dissociate the enzyme from the substrate.
   11. To prevent star activity, make sure that you use the recommended buffer, that the amount of glycerol in the reaction is no more than 5% of the total reaction volume

1. Excise the DNA fragment from agarose gel with clean, sharp scalpel.
2. Weigh the gel slice. Add 3 volumes buffer QG to 1 volume of gel( 100 mg gel = 100 ul). ( if we have 18 mg of gel take around 20 ul of buffer QG)
3. Incubate at 50 degree celsius for 10 minutes. Vortex after 5 minutes to dissolve the gel slice . After gel has dissolved the color of mixture is yellow. NOTE: tilt the MCT upside down, to see if the gel has completely dissolved. If it has not dissolved, incubate it for 2-3 minutes more. NOTE: if mixture color is orange or violet, ad 10 ul 3M sodium acetate, pH 5.0 and mix. The mixture turns yellow.
4. Add 1 gel volume isopropanol to sample and mix.
5. Take the whole volume in the MCT and transfer it to spin column.
6. Spin it on minispin for 90 sec at maximum speed.
7. Discard the flow through
8. Add 750 ul of PE buffer. in the spin column.
9. Spin it on minispin for 90 sec at maximum speed.
10. Discard the flow through and centrifuge once again for 90 seconds at maximum speed.
11. Place QIAquick column in 1.5 mL fresh MCT.
12. Add EB buffer through the QIA quick column center. ( around 20 – 25 ulL depending on application)
13. Spin for 2 minutes at maximum speed in minispin.

id="pfont" Annealing Temperature 54.5 degC