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Trouble with overlap extension pcr - (Jan/10/2014 )

Hello, i am trying to get a point mutation using overlap extension pcr.

The first round of pcr work excelent, i get my 2 bands, from external primer foward to mutagenic primer reverse, and from mutagenic foward to external reverse.

The problem is in the second one, the reaction just can't work properly!.

I can't get the third band between the two external primers, i only get the two bands mentioned before.

i tried with temperature ramp from annealing and magnesium ramp, but i get only the same two bands, the third band doesn't appeared!.

 

i tried to give more denaturation time in the pcr cycles, but when i did it, i don´t have any amplification  (and here i have another question... if i give a lot of time of denaturation (i gave it 5 minutes), why reaction doesn't work? even the control doesn´t amplified ).

 

thermal cycling conditions : initial denaturation 10 minutes to 95 °C, denaturation 45 sec. to 95°C, annealing 60 sec. to 50°C, extension 60 sec. to 72°C, and a final extension 10 minutes to 72°C x 25 cycles

 

I just can't understand why it doesn't work, i use the same protocol in other point mutation, and it works great.

 

I pray for help

 

Thanks in advance

-Guierdy Katherine-

In overlap extension, you should not see the two bands of the products you are combining, ever. If this is the case, you are using way too much template PCR product for your PCR reactions. Also, you are only using the forward primer of piece #1 and the reverse primer of piece #2 right?

 

I cant think of a scenario where you would generate those two products with only the external primers. Thus, you have either added all 4 primers, or you are using near microgram quantities of each PCR product as template.

-HOYAJM-

In overlap extension, you should not see the two bands of the products you are combining, ever. If this is the case, you are using way too much template PCR product for your PCR reactions. Also, you are only using the forward primer of piece #1 and the reverse primer of piece #2 right?

 

I cant think of a scenario where you would generate those two products with only the external primers. Thus, you have either added all 4 primers, or you are using near microgram quantities of each PCR product as template.

thanks for reply, i used only the external primers,and the quantities of each PCR product as template, i always used the same quantity, and  get the same two bands of the first PCR, but also, i always get the third with the combination of both products

-Guierdy Katherine-

 

In overlap extension, you should not see the two bands of the products you are combining, ever. If this is the case, you are using way too much template PCR product for your PCR reactions. Also, you are only using the forward primer of piece #1 and the reverse primer of piece #2 right?

 

I cant think of a scenario where you would generate those two products with only the external primers. Thus, you have either added all 4 primers, or you are using near microgram quantities of each PCR product as template.

thanks for reply, i used only the external primers,and the quantities of each PCR product as template, i always used the same quantity, and  get the same two bands of the first PCR, but also, i always get the third with the combination of both products

 

If that is the case, then your external primers are not stringent enough. They are binding in multiple places to give those additional bands

-HOYAJM-