PCR amplification of differential DNA
PCR amplification of differential DNA
In the reactions described in this protocol, differential DNA is selectively amplified. There should be at least 4 reactions per experiment: 1) ablated test DNA; 2) unablated detector control (1-c); 3) reverse ablated test DNA; 4) reverse unablated control (2-c). This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
PCR amplification of differential DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
1. Buffers, solutions and reagents
dNTP solution (contains all 4 dNTPs, 10 mmol/L each)
2. enzymes and enzyme buffers
PCR reaction buffer, 10X
Polymerase mix, 50X (Advantage2, Clontech, or equivalent)
3. Nucleic acids and oligonucleotides
DNA samples (i.e., each of the ablation samples in step 16 of Option 2)
Nested PCR primer NP1 (10umol/L)
Nested PCR Primer NP2R (10umol/L)
PCR Primer PI(10umol/L)
Unabated detector control from Step 8 of Phase 4 of Protocol 1
4. Specialized equipment
Thermal cycler, preset to 72°C
5. Additional reagents
Reagents and equipment required for 2% agarose gel electrophoresis
II. Methods
1. Primary PCR
(1) For each diluted DNA sample (i.e., each ablated sample in Step 16 of Protocol 2, and the corresponding diluted but unablated detector control in Step 7 of Phase 4 of Protocol 1), dispense 1ul of each into accurately labeled centrifuge tubes. Frozen samples should be thawed and incubated at 72°C, then blown up and mixed before use.
(2) Take another centrifuge tube and prepare a body mix for all primary PCR tubes. Mix the reagents in the following order.
Sterilized water 19.5ul
PCR reaction buffer, 10X 2.5ul
dNTP solution (10mmol/L) 0.5ul
PCR Primer PI (10pmol/L) 1.0ul
50X polymerase mix 0.5ul
Total Volume 24.0ul
(3) Take 24ul of Master Mix and dispense into each reaction tube prepared in step 1.
(4) Cover with 1 drop of mineral oil.
(5) In a thermal cycler, incubate the reaction mixture at 74°C for 5 min to extend the junction. This step "fills in" the missing strand of the junction and thus creates the binding site for the PCR primer. Do not remove the sample from the thermocycler.
(6) Start the following cycle immediately. 
(7) Take 4ul from each tube and analyze on a 2.0% agarose TAE gel.
For some complex ablations (complex tissues or their nuclear genes tantalum), it is recommended to perform the Primary PCR in two steps, which significantly reduces background. First, perform the primary PCR as described in this protocol, and then perform another primary PCR as described in Step 10 of Phase 1 of Protocol 4, followed by a secondary PCR (Protocol 3).
2. Secondary PCR
(8) Dilute 2ul of each primary PCR product in 38ul of water.
(9) Add lul of each primary PCR product diluted in step 1 to a labeled centrifuge tube.
(10) Mix the reagents in the following order to prepare a master mix for the secondary PCR and an additional reaction.
Sterilized water 18.54ul
PCR reaction buffer, 10X 2.5ul
Nested PCR primer NH (10umol/L) 1.0ul
Nested PCR Primer NP2R (10umol/L) 1.0ul
dNTP solution (10 mmol/L) 0.5ul
50X polymerase mix 0.5ul
Total Volume 24.0ul
(11) Dispense 24ul of Master Mix into each of the reaction tubes from step 2.
(12) Cap with 1 drop of mineral oil.
(13) Start the following cycle immediately. 
(14) Take 4ul from each tube and analyze on a 2.0% agarose TAE gel.
(15) The reaction product should be stored at -20°C. This PCR product is now enriched for differential DNA.
If MOS is not required, you can proceed directly to the section on "Cloning of ablated DNA".