1. Perform the necessary calculations for setting up the AB and CD PCRs. Notes f
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Question
1. Perform the necessary calculations for setting up the AB and CD PCRs. Notes for calculations: Final GoTaq reaction buffer should be 1x Final MgCl2 concentration should be 3 mM Final dNTP concentration should be 200 uM Use 1 L of purified rDNA (make sure you use rDNA that contains the fragment that corresponds to your gene of interest) Final primer concentration should be 1 HM (each) Use 5 U GoTaq per reaction Total volume should be 50 L ab PCR cd PCR 5x GoTaq reaction buffer 25 mM MgCl2 10 mM dNTP mix 1 uL of purified rDNA 10 uM fwd primer 10 uM rev primer 5 Units/ul GoTaq Sterile water Total 50 ul 50 ulExplanation / Answer
ab PCR
cd PCR
5x GoTaq reaction buffer
10 µl (1X)
10 µl (1X)
25mM MgCl2
6 µl (3 mM)
6 µl (3 mM)
10mM dNTP mix
1 µl (200 µM)
1 µl (200 µM)
1 µl of purified DNA
1 µl
1 µl
10 µM Forward Primer
5 µl (1 µM a primer)
5 µl (1 µM c primer)
10 µM Reverse Primer
5 µl (1 µM b primer)
5 µl (1 µM d primer)
5 Units/µl GoTaq
1 µl
1 µl
Sterile water
21 µl
21 µl
Total
50 µl
50 µl
ab PCR
cd PCR
5x GoTaq reaction buffer
10 µl (1X)
10 µl (1X)
25mM MgCl2
6 µl (3 mM)
6 µl (3 mM)
10mM dNTP mix
1 µl (200 µM)
1 µl (200 µM)
1 µl of purified DNA
1 µl
1 µl
10 µM Forward Primer
5 µl (1 µM a primer)
5 µl (1 µM c primer)
10 µM Reverse Primer
5 µl (1 µM b primer)
5 µl (1 µM d primer)
5 Units/µl GoTaq
1 µl
1 µl
Sterile water
21 µl
21 µl
Total
50 µl
50 µl
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