PROTOCOLS FOR EXPERIMENT 3 (Labs 6, 7, 8, 9, 10 and 11)

Lab 6: Restriction digestion of pScr and pUC19 and gel isolation.

Set up the following restriction digests:

Digest the DNA for 1 hour at 37 C.

To reaction 1 add 5 ul of 5X sample loading buffer and load 25 ul on an agarose gel.

From reaction 2 remove 1 ul of the reaction and mix with 5 ml of 5X sample loading buffer and load on the gel.

Run a 20 ul sample of diluted uncut DNA supplied to you.

Load some DNA ladder on the gel.

Processing the vector

To the remainder of reaction 2 add 20 ul of phenol/CHCl3 (1:1 v:v) and vortex the sample. Centrifuge the sample for 10 min at 13,000 rpm in a microcentrifuge.

REMEMBER TO BALANCE YOUR TUBE!!

Remove the upper phase and place in a fresh microcentrifuge tube. Add 2 ul of 3M NaAcetate pH 5.4 to the sample and then add 50 ul of absolute (100%) ethanol, vortex. Place in freezer for 20 minutes and centrifuge for 10 min at 13,000 rpm.

REMEMBER TO BALANCE YOUR TUBE!!

Remove the ethanol mixture with a pippetor making sure you do not disturb the pellet on the side of the tube. Add 50 ul of 70% ethanol to the tube gently and remove. Leave the tube out to air dry. When the DNA is dry, add 20 ul of H2O. Store sample in freezer until next lab.

DNA fragment isolation

Take a picture of the gel and save the file. If you successfully digested the pScr DNA proceed to the next step.

You will be using a QIA quick Gel Extraction Kit.

1.) Excise the DNA fragment from the agarose gel with a razor blade. Make sure that you are wearing gloves and the face shield while working exposed to the UV transilluminator. Cut out as small of a piece as possible. Place agarose peice in a microcentrigfuge tube. Estimate its volume by quickly spinning the agarose in a microcentrifuge. Look on the side of the tube you will find marks for 100 ul etc. The microcentrifuge tubes are graduated.

2.) Add 3 volumes of Buffer QG to 1 volume of gel.

3.) Incubate at 50 C for 10 min (or until the gel slice has completely dissolved). To help dissolve the gel, mix by vortexing the tube every 2-3 min during the incubation.

4.) After the gel slice has dissolved completely, check that the color of the mixture is still yellow.

5.) Add 1 gel volume of isopropanol to the sample and mix.

6.) Place a QIAquick spin column in a provided 2 ml collection tube (will be assembled already).

7.) To bind the DNA, apply the sample to the QIAquick column, and centrifuge for 1 min at 7,000 rpm in a microcentrifuge.

REMEMBER TO BALANCE YOUR TUBE!!

The maximum volume that can be loaded into the column is 800 ul; if your total volume is greater than 800 ul simply load the column again with the rest and centrifuge again.

8.) Discard the flow-through and place QIAquick column back in the same collection tube.

9.) To wash, add 0.75 ml of Buffer PE to the QIAquick column and centrifuge for 1 min at 7,000rpm.

REMEMBER TO BALANCE YOUR TUBE!!

10.) Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 7,000 rpm.

REMEMBER TO BALANCE YOUR TUBE!!

11.) Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

12.) To elute DNA add 50 ul of H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm.

REMEMBER TO BALANCE YOUR TUBE!!

13.) Store the sample in the freezer until next week

Lab 7: Ligation of DNA.

Set up the following ligation reactions.

Incubate at room temperature for 1 hour and put in the freezer on your rack.

Lab 8: Transformation of DH5a cells

Goal: to transform competent E. coli cells with ligated DNA.

REMEMBER: All LIVE bacterial waste must be placed in the BIOHAZARD waste container.

1.) Place the two microcentrifuge tubes on ice and add 50 ul aliquot of thawed DH5a cells to each tube gently mixing the cells with the sterile pipet tip prior to aliquoting.

2.) Add 2ul of each ligation reaction in a sterile microcentrifuge tube containing the cells. DO NOT MIX BY PIPETTING UP AND DOWN. Gently stir the cells and DNA with pipet tip.

3.) Incubate the microcentrifuge tubes on ice for 30 minutes.

6.) Heat shock the cells for 20 seconds in a 42 degrees Celsius water bath without shaking.

7.) Place tubes on ice for 2 minutes.

8.) Add 1 ml of LB liquid media.

9.) Incubate the microcentrifuge tubes at 37 degrees Celsius for 1 hour.

10.) Spin down the tubes at 10000 rpm for 30 seconds. Dump out the supernatant into the BIOHAZARD waste.

11.) Add 100 ul of LB media to the cells and resuspend by GENTLY pipetting up and down a couple of times.

12.) Spread the whole volume of cells onto an LB+Amp+X-gal plate.

11.) Put tubes in the bacterial waste container for autoclaving.

12.) Incubate plates overnight upside-down at 37 degrees Celsius. The technician will remove the plates the next day and put them in the fridge.

13.) One group in each lab section will transform a control.

Agarose gel of ligations and DNA fragment isolation

Add 2.5 ul of 5X sample buffer to 10ul of ligation reactions 1 and 2, and the isolated DNA fragment. Load these three samples on an agarose gel with a lane containing size markers.

Lab 9: Picking a colony for miniprep DNA isolation

Retrieve your transformation plates and document the results. What colour are the colonies on each plate? Why? Also find out the results from the control plate that was transformed for your section.

If possible, pick a white colony from the reaction 2 transformation plate using the loop provided (remember to sterilize it by flaming). Innoculate 2 ml of sterile liquid LB + Amp broth provided.

Label your tube clearly. Leave at room temperature for 1 hour and place in the rack in the fridge. They will be grown and ready for you for the next lab.

Lab 10: Miniprep DNA isolation

REMEMBER: All LIVE bacterial waste must be placed in the BIOHAZARD waste container.

Goal: To prepare plasmid DNA to verify the insertion of the Scr DNA fragment into pUC 18.

It is extremely important that you label the tubes clearly. Two marks will be deducted for unclear labeling.

The tube that you inoculated last week will be be placed at 37 C overnight in a shaker by the technician. Be sure to label your tubes.

1. Remove approximately 1.5 ml of the culture with a transfer pipette and put into a labeled 1.5 ml microcentrifuge tube. Squirt some bleach into the culture tube to kill the bacteria (about 2 ml is sufficient) and then place the culture tube containing bleach in the labeled rack by the sink. DO NOT pour into biohazard waste.

2. Centrifuge the cells at full speed in a Microcentrifuge at full speed for 1 minute.

REMEMBER TO BALANCE YOUR TUBE!!

3. Remove supernatant with pipettor or pour off the supernatant. The supernatant must go into the biohazard waste beaker provided.

4. To resuspend pelleted bacterial cells: Add 250 ul of Buffer P1, close the tube and vortex using the vortex mixer. Alternatively, leave the tube open and using the pipettor pull the pellet into the pipet tip and back out several times until cells are dispersed. Look to see that the pellet has disappeared and that there are no cell clumps. Tips must go into biohazard waste since cells are not yet lysed.

For maximum yield, the cells need to be well dispersed.

5. To lyse the cells: add 250 ul Buffer P2, close the lid and gently invert the tube 4-6 times. To invert a tube hold the tube between your thumb and index finger and gently rock the tube back and forth.

*From this point onwards, waste can be placed into non-hazardous waste unless specified otherwise.

It is important that you do not vortex the tube because this will shear the genomic DNA. It is important not to let the lysis reaction proceed for more than 5 minutes.

LyseBlue reagent was added to the P1 buffer, and when P2 buffer is added to the miniprep, a blue colour should develop. If you have resuspended the cell properly and mixed the solution well, the solution should be a uniform blue colour. If the solution contains brownish cell clumps or colourless areas, continue mixing gently until a uniform blue colour is achieved.

6. To neutralize: add 350 ul Buffer N3. Quickly close the tube and gently invert 4-6 times. Leave on ice for 10 minutes.

The solution should become cloudy. The addition of buffer N3 changes the LyseBlue reagent from blue to colourless; therefore, if you have mixed Buffer N3 well the whole solution should be colourless. If blue colour still remains continue to gently mix the contents of the tube.

7. Centrifuge at full speed in a Microcentrifuge for 10 minutes.

REMEMBER TO BALANCE YOUR TUBE!!

After centrifugation there should be a white pellet and a clear supernatant.

8. Binding DNA to the column: the kit supplies a column that is inserted into a large (2ml) microcentrifuge tube; label the tube and use it to collect the flow through. Using a pipettor remove the supernatant and apply it to the top of the column. Centrifuge for 30-60 seconds in a Microcentrifuge .

REMEMBER TO BALANCE YOUR TUBE!! Use a microcentrifuge tube filled with 1.5 ml of water.

9. Discard the flow through in the microcentrifuge tube. Use the waste beaker provided.

10. To wash the column: add 0.5 ml Buffer PB and centrifuge for 30-60 seconds in a Microcentrifuge .

REMEMBER TO BALANCE YOUR TUBE!! Use a microcentrifuge tube filled with 1.5 ml of water.

11. Discard the flow through. Use waste beaker provided.

12. To wash the column a second time: add 0.74 ml of Buffer PE and centrifuge for 30-60 seconds in a Microcentrifuge .

REMEMBER TO BALANCE YOUR TUBE. Use a microcentrifuge tube filled with 1.5 ml of water.

13. Discard the flow through. Use waste beaker provided.

14. To remove residual wash buffer: centrifuge for 1 minute in a Microcentrifuge .

15. To elute the DNA from the QIAprep column: place the QIAprep column in a clean, labeled 1.5 ml microcentrifuge tube. Add 50 ul of water to the center of the QIAprep column and let stand for 1 minute. Centrifuge for 1 minute in a Microcentrifuge .

REMEMBER TO BALANCE YOUR TUBE. Use a microcentrifuge tube with 1 ml of water.

16. You should have about 50 ul of a DNA solution in the bottom of the tube. Return your rack to the TA containing this labeled tube.

Lab 11: Restriction enzyme digestion

You will need to set up an EcoR1/HindIII double digest with 5 ul of DNA and run an Agarose Gel and record the data using Gel documentation as you have performed previously in the introductory lab. Run lanes with uncut DNA as well.

IMPORTANT NOTICE: As part of the preparation for this lab, you are required to have a table of volumes for the reactions written in your lab books before entering the lab.