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PCR SOEing
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PCR SOEing (Polymerase Chain Reaction - Splicing by Overlapping Extension) is a technique we've begun using in our lab for the first time this year. Two or more linear DNA fragments can be attached together without numerous steps of restriction digestion and ligation. 3' primers are created for each fragment which match the overlap 3' regions on the next downstream fragment, and 5' primers are created for each fragment which match the overlap 5' region on the previous upstream fragment. | PCR SOEing (Polymerase Chain Reaction - Splicing by Overlapping Extension) is a technique we've begun using in our lab for the first time this year. Two or more linear DNA fragments can be attached together without numerous steps of restriction digestion and ligation. 3' primers are created for each fragment which match the overlap 3' regions on the next downstream fragment, and 5' primers are created for each fragment which match the overlap 5' region on the previous upstream fragment. | ||
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==PCR SOEing Protocol== | ==PCR SOEing Protocol== | ||
For the process to work, equal amounts of molecules of all fragments are required. The calculations we used for finding these amounts are as follow : | For the process to work, equal amounts of molecules of all fragments are required. The calculations we used for finding these amounts are as follow : | ||
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[concentration of fragment #1]*[length of fragment #1 in bp]*[volume of fragment #1] = [concentration of fragment #2]*[length of fragment #2 in bp]*[volume of fragment #2] | [concentration of fragment #1]*[length of fragment #1 in bp]*[volume of fragment #1] = [concentration of fragment #2]*[length of fragment #2 in bp]*[volume of fragment #2] | ||
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Equal amounts of the oligos are then mixed with the DNA fragments along with PCR Supermix and placed into a Thermocycler under the following protocol: | Equal amounts of the oligos are then mixed with the DNA fragments along with PCR Supermix and placed into a Thermocycler under the following protocol: | ||
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| - | Step1: 95C for 5 min. | + | |
| - | Step2: 95C for 15 sec. | + | Step1: 95C for 5 min. |
| - | Step3: [lowest annealing temperature of all the primers] for 60 sec. | + | |
| - | Step4: 68C for [1 minute plus 1 minute for every 1kb of the total PCR product]. | + | Step2: 95C for 15 sec. |
| - | Repeat Step 2: 35 times. | + | |
| - | Step6: Hold at 4C. | + | Step3: [lowest annealing temperature of all the primers] for 60 sec. |
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| + | Step4: 68C for [1 minute plus 1 minute for every 1kb of the total PCR product]. | ||
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| + | Repeat Step 2: 35 times. | ||
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| + | Step6: Hold at 4C. | ||
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At the end of the process, the resulting DNA must be run through gel electrophoresis to ensure the correct products were formed. | At the end of the process, the resulting DNA must be run through gel electrophoresis to ensure the correct products were formed. | ||
Revision as of 04:30, 29 October 2008
PCR SOEing (Polymerase Chain Reaction - Splicing by Overlapping Extension) is a technique we've begun using in our lab for the first time this year. Two or more linear DNA fragments can be attached together without numerous steps of restriction digestion and ligation. 3' primers are created for each fragment which match the overlap 3' regions on the next downstream fragment, and 5' primers are created for each fragment which match the overlap 5' region on the previous upstream fragment.
PCR SOEing Protocol
For the process to work, equal amounts of molecules of all fragments are required. The calculations we used for finding these amounts are as follow :
[concentration of fragment #1]*[length of fragment #1 in bp]*[volume of fragment #1] = [concentration of fragment #2]*[length of fragment #2 in bp]*[volume of fragment #2]
Equal amounts of the oligos are then mixed with the DNA fragments along with PCR Supermix and placed into a Thermocycler under the following protocol:
Step1: 95C for 5 min.
Step2: 95C for 15 sec.
Step3: [lowest annealing temperature of all the primers] for 60 sec.
Step4: 68C for [1 minute plus 1 minute for every 1kb of the total PCR product].
Repeat Step 2: 35 times.
Step6: Hold at 4C.
At the end of the process, the resulting DNA must be run through gel electrophoresis to ensure the correct products were formed.
