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PCR of ADA Mutations
Goals
- Prepare ADA primers in order to conduct a polymerase chain reaction (PCR)
- Run HRP Luminol Assay
Preparation
- Assigned Primer= ADA K110A F (forward primer)
- Sequence Issue number: 87635082
- Sequence: ATT TGG CCA AAT GGG CAG TTA TTG AA
- TM= 65.5°C
- MW= 16833.0 g/mol
- 33.4 nm= .56 mg ( amount of present plasmid)
Concentration of plasmid needed for experiment= 100ng/μL
.56mg= 5600 ng
M1V1= M2V2
5600 ng/μL x V1= 100ng x 1000μL
- (The container containing the plasmid only had room for 1mL of water to be added- as a result, a dilution needed to be done in order to get the appropriate concentration.
V2= 17.857μL is the amount of plasmid solution pipetted out in order to get a concentration of 100ng/μL
Procedure
- 1mL of deionized water was added to .56mg of ADA K110A F
- 17.86μL of this solution was pipetted out and added to a centrifuge tube. 982.14μL of autoclaved water was then added to this sample in order to dilute it to 100ng/μL.
- Next, 2 50μL solutions were made with the following components in microcentrifuge tubes. The DNA polymerase was added last.
| Component
|
Volume [μL]
|
| Distilled water |
40.6
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| 10X cloned Pfu buffer |
5
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| 25 mM dNTPs |
0.4
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| 100 ng/μL DNA template |
1
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| Forward primer (K110 f) |
1
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| Reverse primer (K110A r) |
1
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| 2.5 U/μL PfuTurbo DNA polymerase |
1
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- Immediately after the addition of the DNA polymerase, the two sample solutions were placed in a thermocycler. The heating and cooling cycle of the thermocycler for the samples is as follows.
- Heat for 2 minutes at 95°C.
- Heat for 30 seconds at 95°C.
- Heat for 30 seconds at 57°C.
- Heat for 8 min at 72°C.
- Repeat steps two through four 30 times.
- Heat for 10 minutes at 72°C.
- Chill at 0°C until the samples needed again.
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