PCR 2

From FreeBio

Aim

    The purpose of this experiment was to clarify results obtained in the previous experiment.  Both this experiment and the previous experiment, PCR 1, amplified the region surrounding the cat gene by using PCR.  

"Methods"

    In one PCR tube, 81 µL of distilled water, 4 µL of MgSo4, 2µL of dNTPs, 10 µL of HIFI buffer, 1 µL of platinum taq. polymerase, 2 µL of the old forward primer, and 2 µL of the old reverse primer.  After mixing, 51 µL of the solution was transferred into a new PCR tube.  1 µL of the old template was added to one of the PCR tubes while 1 µL of water was added to the second PCR tube.  The tube without template served as the negative control.
    In a new PCR tube, 79 µL  of distilled water, 4 µL of MgSo4, 2µL of dNTPs, 10 µL of HIFI buffer, 1 µL of platinum taq. polymerase, 2 µL of the new forward primer, and 2 µL of the new reverse primer.  50 µL of the mixture was transferred to a new PCR tube.  1 µL of new cat template was added to the first PCR tube and 1 µL of water was added to the second PCR tube.
    The four PCR samples were placed in the PCR machine and cycled as follows:

Step Temperature (ºC) Time

1 95 15’

2 94 30’’

3 56 30’’

4 68 1.5’

5 Go to step 2 7 x

6 94 30’’

7 65 30’’

8 68 1.5’

9 Go to step 6 30x

10 68 5’

11 4 Forever

    After PCR was complete, the products were run on an E-gel with a 2kb ladder and vizualized with UV light.

'Results

   (picture to be uploaded)
    Figure 1: Analysis of PCR Products"  Lanes 6 and 9 contain the 2kb ladder.  Lane 4 contains the old primers and no template while lane 5 contains the old primers and the old template.  Lane 7 contains the new primers and no template while lane 8 contains the new primers and the new template.  Lanes 1-3 and 10-12 contain distilled water.

    The results from this experiment are strange.  A band appears in each lane regardless of whether or not the template was present.  The bands appear to be the correct length of the cat gene and this indicates that somehow, both negative controls were contaminated with cat template.  Perhaps another PCR experiment is necessary.