Molecular Biology Lab 312L

Outline: Experiment 22

1. Isolation of Cheek Cell DNA

We will follow the protocol in the lab manual for isolation of our cheek cell DNA. Note the following hints:

STEP
#4) spin the cheek cell/saline solution at 1500 x g for 10 minutes.

#5) the cell pellet may still be loose. If you can't safely pour off the saline, transfer the remaining cell/saline solution to eppendorf tube(s) and spin in the microfuge to pellet. Then use a pipettor to draw off the supernatant.

#6) everyone should resuspend the chelex solution before they remove an aliquot. The chelex settles out of solution very quickly.

#9) use the special locking eppendorf tubes for the boiling water bath. Use tongs to remove the boat from the boiling water and insert your tube.
 

<>2. Set up PCR Reaction

PCR reactions are often initiated by adding either magnesium (a cofactor of Taq Polymerase) or by adding the template DNA. The Taq and other components make up a "master mix" that is aliquotted into each tube.

For this experiment, each student will have a 40 ml reaction volume containing:

dH2O
15 ml
cheek cell DNA
5 ml
MgCl2
(25 mM stock)
3 ml
master mix
17 ml
TOTAL
40 ml

The master mix has the components listed below. We must make enough for everyone in the class. See if you can do the calculations.
 

17 ml of master mix in a total reaction volume of 40 ml
needed per student:  times (the # of students) + (2 extra) = 
10 x Buffer B = 4 ml  
forward primer  = 2.5 ml  
reverse primer = 2.5 ml  
^10 mM dNTP's = 1 ml  
*Taq Polymerase = 
(5U/ml, see below)		  
"dH2O =   
TOTAL = 17 ml TOTAL = 

* recommended concentration of Taq is 0.02 - 0.03 U/ml for the total volume.
The total volume would be (the number of students) x (40 ml reaction volume per student). So, for 10 students, the total volume is 400 ml. The Taq has 5 U/ml. How much Taq Polymerase do we need to have 0.02 - 0.03 U per ml in our 400 ml?
    To calculate:
                                X ml = (0.02 U)(total PCR volume)
                                                                5 U/ml
 

^The dNTP's are present at 10 mM per NTP, or 40 mM total conc.

" Add enough water to bring the total volume to 17 ml.
 

3. Initiate the PCR Reaction

Aliquot dH2O and your cheek cell DNA into the PCR tube.
Set up an "assembly line" and do the following in sequence:

When all tubes are finished, start the PCR reaction. The PCR parameters are given in the lab book, and are linked to a soak file.