PCR reactions

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1.

We set up our first gradient PCR reaction to determine the optimal annealing temperature for each of our genes.

• We centrifuged and deluted 10 μL of our 100μM reverse and forward primer solution to 100μL with TE Buffer.
• We dissolved our 500ng of otimized HNMT in 1000μL TE Buffer then we vortexed and centrifuged the solution.
• We dissolved our 500ng of wild type HNMT in 1000μL TE Buffer then we vortexed and centrifuged the solution.
• We dissolved our 1000ng of otimized DAO in 1000μL TE Buffer then we vortexed and centrifuged the solution.
• We dissolved our 1000ng of wild type DAO in 1000μL TE Buffer then we vortexed and centrifuged the solution.

We added the following substrates in order:

Component	Final concentration	50-μL rxn
2X Platinum TM SuperFi TM II PCR Master Mix	1X	25 μL
Forward primer	0.5 μM	3 μL
Reverse primer	0.5 μM	3 μL
Template DNA	0.1–10 ng plasmid (5-100 ng genomic DNA)	2 μL
Water, nuclease free	-	17 μL

We ran the following PCR program:


We placed our PCR tube into the maschine in the following order:
A: O1 and W1
B: O1 and W1
C: -
D: O2 and W2
E: O2 and W2
F: -
G: O3 and W3
H: O3 and W3

We added the 6X DNA Loading dye to each PCR tubes marked in red above. We loaded the wells of the gels with 12μL of HNMT solution in the following order:

1. O1
2. O2
3. O3
4. Ladder (8 μL)
5. W1
6. W3

We loaded the wells of the gels with 12μL of DAO solution in the following order:

1. O1
2. O2
3. O3
4. Ladder (8 μL)
5. W1
6. W3

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2.

Problem: few PCR products were generated
Solution: We decided to change the volume of the primer - and the DNA solutions added to the PCR reaction, to produce more DNA.
We added the following substrates in order:

Component	Final concentration	50-μL rxn
2X PlatinumTM SuperFiTM II PCR Master Mix	1X	25 μL
Forward primer	0.5 μM	2 μL
Reverse primer	0.5 μM	2 μL
Template DNA	0.1–10 ng plasmid (5-100 ng genomic DNA)	3 μL
Water, nuclease free	-	18 μL

We added the 6X DNA Loading dye to each PCR tubes.
We loaded the wells of the gels with 12μL of HNMT solution in the following order:

1. TO1
2. TO2
3. TO3
4. Ladder
5. GO1
6. GO2
7. GO3
8. GW1
9. GW2

We loaded the wells of the gels with 12μL of DAO solution in the following order:

1. -
2. TW1
3. TW2
4. TW4
5. Ladder
6. TO1
7. TO2
8. TO3
9. -

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3.
HNMT

Problem: few PCR products were generated
Solution: We raised the MgCl2 concentration
We ran more cycles and set them longer
We used 3 different Master Mix, containing different Taq polimerases.
We added the following substrates in order: (each optimized and wild type)

	S1(O/W)	S2(O/W)
SuperFi Master Mix (2X)	14 μL	14 μL
Forward primer	2 μL	2 μL
Reverse primer	2 μL	2 μL
HNMT gene	8 μL	8 μL
Water, nuclease free	16 μL	20 μL
10X Dream Taq Buffer	8 μL	4 μL
Total	50 μL	50 μL

	T1(O/W)	T2(O/W)
DreamTaq Master Mix (2X)	14 μL	14 μL
Forward primer	2 μL	2 μL
Reverse primer	2 μL	2 μL
HNMT gene	8 μL	8 μL
Water, nuclease free	16 μL	20 μL
10X Dream Taq Buffer	8 μL	4 μL
Total	50 μL	50 μL

	N1(O/W)	N2(O/W)
NED Master Mix (2X)	14 μL	14 μL
Forward primer	2 μL	2 μL
Reverse primer	2 μL	2 μL
HNMT gene	8 μL	8 μL
Water, nuclease free	16 μL	20 μL
10X Dream Taq Buffer	8 μL	4 μL
Total	50 μL	50 μL

We ran our PCR reaction for HNMT:

We added the 6X DNA Loading dye to each PCR tubes.
We loaded the wells of the gels with 12L of optimized HNMT solution in the following order:

1. -
2. S1O
3. S2O
4. T1O
5. Ladder
6. T2O
7. N1O
ű
8. N2O
9. -

We loaded the wells of the gels with 12μL of wild type HNMT solution in the following order:

1. -
2. S1W
3. S2W
4. T1W
5. Ladder
6. T2W
7. N1W
ű
8. N2W
9. -

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4.
DAO

Problem: unnecessary large fragments in PCR products
Solution: We decreased the MgCl2 concentration
We annealing time for 20s.
We added more EDTA.
We added the following substrates in order: (each optimized and wild type)

	D31 (O/W)	D32 (O/W)	D33 (O/W)	D34 (O/W)
MM (1X)	25 μL	25 μL	25 μL	25 μL
Forward primer	1 μL	1 μL	1 μL	1 μL
Reverse primer	1 μL	1 μL	2 μL	1 μL
DAO gene	4 μL	4 μL	4 μL	4 μL
Water, nuclease free	14 μL	17 μL	12 μL	9 μL
EDTA	5 μL	2 μL	5 μL	10 μL
Total	50 μL	50 μL	50 μL	50 μL

We ran our PCR reaction for DAO:

We added the 6X DNA Loading dye to each PCR tubes.
We loaded the wells of the gels with 12μL of DAO solution in the following order:

1. D31O
2. D32O
3. D33O
4. D34O
5. Ladder
6. D31W
7. D32W
8. D33W
9. D34W

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5. and 6.
HNMT

According to the previous PCR reaction we decided to use S1 and N1.
We added the following substrates in order: (each optimized and wild type)

	S1(O/W)
SuperFi Master Mix (2X)	14 μL
Forward primer	2 μL
Reverse primer	2 μL
HNMT gene	8 μL
Water, nuclease free	16 μL
10X Dream Taq Buffer	8 μL
Total	50 μL

	N1(O/W)
NED Master Mix (2X)	14 μL
Forward primer	2 μL
Reverse primer	2 μL
HNMT gene	8 μL
Water, nuclease free	16 μL
10X Dream Taq Buffer	8 μL
Total	50 μL

We set up previously used PCR reaction

DAO

According to the previous PCR reaction we decided to use D31 and D32.

	D41(O/W)	D42(O/W)
MM (1x)	25 μL	25 μL
Forward primer	1 μL	1 μL
Reverse primer	1 μL	1 μL
DAO gene	4 μL	4 μL
Water, nuclease free	14 μL	17 μL
EDTA	5 μL	2 μL
Total	50 μL	50 μL

We set up previously used PCR reaction.
We added the 6X DNA Loading dye to each PCR tubes. We loaded the wells of the gels with 12μL of DNA solution in the following order:

1. S1O
2. S1W
3. N1O
4. N1W
5. Ladder
6. D310
7. D31W
8. D32O
9. D32W

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7. and 8.
HNMT

According to the previous PCR reaction we decided to use S1.

	S1(O/W)
SuperFi Master Mix (2X)	14 μL
Forward primer	2 μL
Reverse primer	2 μL
HNMT gene	8 μL
Water, nuclease free	16 μL
10X Dream Taq Buffer	8 μL
Total	50 μL

DAO

According to the previous PCR reaction we decided to use D31.

	D41(O/W)
MM (1x)	25 μL
Forward primer	1 μL
Reverse primer	1 μL
DAO gene	4 μL
Water, nuclease free	14 μL
EDTA	5 μL
Total	50 μL

We set up previously used PCR reaction
We digested our DNA inserts. We loaded the wells of the gel in the following order:
1-3. optimized HNMT
4. ladder
5-6. optimized DAO
7-8. wild type HNMT

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9.

We digested our petDUET plasmid.