PROTOCOLS FOR EXPERIMENT 1 (Labs 2 and 3)
Lab 2: Plasmid DNA preparation
Goal: To prepare plasmid DNA for DNA sequencing.
It is extremely important that you label the tubes clearly. 2 marks will be deducted for unclear labeling.
You will be supplied with a 2 ml culture containing bacteria that carry the EST. At this time you will be provided with the name of the EST. This name shall appear on all labels.
1. Remove 1.5 ml of the culture with transfer pipette and put into a labeled 1.5 ml microcentrifuge tube, or carefully pour the bacteria into the tube filling it up to the 1.5 ml mark of your labeled 1.5 ml microcentrifuge tube. Squirt some ethanol into the culture tube to kill the bacteria (about 2 ml is sufficient). Pour bacteria and ethanol into waste beaker provided.
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 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.
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.
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 (2 ml) 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, or carefully pour the supernatant into 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 3: Excision of EST fragment from vector using restriction enzymes, agarose gel electorphoresis and DNA sequence analysis.
Goal: Determine whether DNA was isolated and determine the size of the cDNA insert.
Each member of the group will set up their own 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. Depending on the group size, there should be four to five lanes loaded on the gel. Lane 1 should contain the ladder; lane 2 should contain uncut plasmid DNA; lane 3-5 should contain EcoR1/HindIII digested plasmid DNA performed by each group member.
REMEMBER TO SAVE YOU IMAGE AS A .TIF FILE (RAW DATA). AND MAKE SURE YOU HAVE SAVED IT TO THE RIGHT FOLDER.
Show the gel to your TA. He/she will advise you on whether the plasmid should be sent for DNA sequencing.
DNA sequencing
You have to dilute your DNA by 2-fold before it is sent for sequencing. In order to do so, take 10 ul of your DNA solution and place in an microcentrifuge tube and add 10 ul of water to the tube.
Give this labeled tube to your TA.
The DNA sequence will be in your results folder.
Gloves, lab coat and safety eyeglasses must be worn ALL times when handling these Buffers for plasmid DNA preparation.
Buffer P1 contains RNase A: sensitizer
Buffer N3 contains guanidine hydrochloride, and acetic acid: harmful irritants.
Buffer P2 contains guanidine hydrochloride: irritant
Buffer PB contains guanidine hydrochloride and isopropanol: harmful, flammable, irritant.
Ethidium bromide: mutagen