Given the information, can someone help me answer this question? 2. Perform the
ID: 212735 • Letter: G
Question
Given the information, can someone help me answer this question? 2. Perform the necessary calculations for setting up the stitch PCR. Notes for calculations: Final GoTaq reaction buffer should be 1x Final MgCl2 concentration should be 3 mM Final dNTP concentration should be 200 HM Use 2 L of each template DNA (AB product and CD product) Final primer concentration should be 1 uM (each) Stitch PCR 5x GoTaq reaction buffer 25 mM MgCl2 10 mM dNTP mix Template: AB product Template: CD product 10 uM fwd primer 10 uM rev primer Sterile water Total 50 uExplanation / Answer
Stock concentration
Final concentration
Volume added to Stitch PCR
5X Go taq reaction buffer
5X
1X
10 microliter
25 mM MgCl2
25 mM
3mM
6 microliter
10mM dNTP
10 mM
200 micromolar
1 microliter
Template AB product
2 microliter
Template: CD product
2 microliter
10 micromolar Forward primer (b primer)
10 micromolar
1 micromolar
1 microliter
10 micromolar reverse primer (b primer)
10 micromolar
1micromolar
1 microliter
Sterile water
27 microliter
Total volume
50 microliter
1) 5X Go Taq reaction buffer:
It is diluted 1: 5 to get 1X from stock of 5X solution.
If final volume is 10 microliters, 10 +40= 50. Hence, 10 microliters of reaction buffer is added.
2) 25 mM MgCl2
Required is 3 mM in 50 microliters
C1V1=C2V2
25 mM X a= 3 mM X 50 microliter
a=6 microliters
3) 10 mM (millimolar) of dNTPS
Required is 200 micromolar dNTP
1 mM= 1000 micromolar
C1V1= C2 V2
10 * 1000 micromolar X a= 200 micromolar X 50 microliter
a= 1 microliter
4) Template AB and CD are added as 2 microliters each
5) 10 micromolar of forward and reverse primer
Required is 1 micromolar
A 1: 10 dilution needs to be performed. Hence, volume added is 1 microliter each.
6) Sterile water = 50 microliter – (sum of all reagents)= 50 –(10+6+1+2+2+1+1)= 50-23= 27 microliter
Stock concentration
Final concentration
Volume added to Stitch PCR
5X Go taq reaction buffer
5X
1X
10 microliter
25 mM MgCl2
25 mM
3mM
6 microliter
10mM dNTP
10 mM
200 micromolar
1 microliter
Template AB product
2 microliter
Template: CD product
2 microliter
10 micromolar Forward primer (b primer)
10 micromolar
1 micromolar
1 microliter
10 micromolar reverse primer (b primer)
10 micromolar
1micromolar
1 microliter
Sterile water
27 microliter
Total volume
50 microliter
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