Feb 02nd 2012
Today Ms. Urban wasn’t in so I worked with Mr. Adams on
another experiment. The previous one, my first experiment didn’t have good
results; in fact, it didn’t work at all, no DNA was copied. So we’re to set up
another one with some adjustments that Ms. Urban made to the “recipe”. This
time we were to use 4 samples of DNA, the same ZAL group 13, 14, 15, and 16.
The first experiment used ZAL 17, 74, 73, 76 and 75. We were also to use the
same part of the DNA, NAD6. The Thermo
cycler program however remained the same QIA42-57.
Before we began to make the master mix, Mr. Adams explained
that they changed some of the recipe so that it matches the “original”
experiment by some Brazilian researchers, he gave the published paper they
wrote for me to get a better idea of what he was talking about.
The changes to the recipe was as follows:
|
PER 1 RXN
|
ORIGINAL AMMOUNT
(microliters)
|
AMMOUNT IN STANDARD
*4(microliters)
|
ALTERED
(microliters) *4
|
|
|
Pure H2O
|
17.5
|
70
|
70
|
|
|
Buffer
|
2.5
|
10
|
10
|
|
|
MgCl2
|
1.5
|
6
|
(2.5)*4=10
|
|
|
dNTPs
|
2.0
|
8
|
8
|
|
|
Primers
|
.5
|
2
|
(.75)*4=3
|
|
|
Polymerase
|
.15
|
.6
|
(.2)*4=.8
|
|
|
TOTAL PER RXN
|
23
|
26.2
|
||
|
DNA
|
2
|
2
|
||
Unfortunately, Mr. Adams and I made a mistake and did not
multiply the given amount by 4 to make the master mix and we ended up with very
little mixture and could only distribute to 2 tubes. The total of the mix
should’ve been about 386.4 microliters for the standard experiment and about
407.2 microliters for the altered mixture. Because there were 32 tubes, 16 for
each group of experiment if we needed to multiply each amount to fit 16 tubes.
Even though we didn’t really make the mixture, I gained a
bit more experience on how to use pipettes and it was great to make mistakes
because it helps me learn and I can be more careful next time. We noted the
mistake and next week we are to work on making the experiment correctly.
