Amplify a ~5kb fragment with 10 different primer pairs, the template DNA is the same, the reaction system is 50ul, and the control has no template and contains a single primer. For reactions with a high number of cycles (25 to 40), a control without template or single primer should be set up. This experiment is from PCR Lab Guide (2nd edition) by Seed Kang and Qu Lijia.
Operation method
Precision amplification experiments for large fragments
Materials and Instruments
Betaine PCR Reaction Buffer TEN+BSA Enzyme and Enzyme Buffer Nucleic Acids and Primers Move I. Materials For more product details, please visit Aladdin Scientific website.
PCR Instrument
1. Buffers, solutions and reagents
The following reagents prepared in sterilized water and stored at the specified temperature conditions.
(1) 5mol/L betaine (SigmaB-2629 or SigmaB-2754)
Filter sterilize and store at room temperature.
(2) 10XKLA PCR reaction buffer, pH 9.2
500 mmol/L Tris base (Sigma; Trizma base)
169 mmol/L(NH )4SO4
25 mmol/LMgCl2
1%Tween20
No adjustment, pH about 9.2 (best for large fragment amplification), for some plasmids, e.g. col El, pH 7.9 is preferred (see Chapter 29); use 2mol/L HCl 10XKLA to pH 7.9, do not insert the pH meter directly into the buffer to avoid contamination with external DNA, but you can take a small drop to test. To avoid freezing of the 10X buffer, filter sterilize and store at 4°C (if the buffer becomes cloudy, shake well before use). For Kletaq LA, the Mg2+ concentration is important. In the full-length Taq enzyme amplification system (10XTLA), MgCl 2 is only 7.5 mmol/L.
(3) TEN+BSA
10 mmol/L Tris-HCl (pH 7.9)
10 mmol/L NaCl
0.lmmol/L EDTA
100ug/ml BSA
-20°C storage.
Generally dilute the mold wrench DNA to 10ng/ml (or less) with TEN+BSA, which increases reproducibility, especially after freeze storage.
2. Enzyme and enzyme buffer
Klentaq LA is 50 to 55 U/ul (recommended enzyme concentration, which varies depending on the length of the replicon; see 6.1 Troubleshooting ②).
3. Nucleic acids and primers
(1) 10/40dNTP mixture
10 mmol/L of each dNTP
40 mmol/LMgCl2
Each dNTP can be purchased as a dry powder from Pharmacia Biotech, dissolved to l0 mmol/L, and stored at -80°C. Generally, dNTP mixes are dispensed in 500/ul portions and stored at 1 80°C, or -20°C if commonly used. dNTP mixes are more conducive to obtaining high yields of PCR products than purchased dNTP (100 mmol/L). The authors did not use freshly purchased dUTP. The authors did not use freshly purchased dUTPase for comparison, so using dUTP may be problematic.
(2) 10umol/L primers
Dilute the primer to l0pmol/L, add 1ul to 50ul of reaction system, and store at -20°C.
4. Equipment
PCR instrument.
Methods
1. Add the following reagents on ice.
10XKLA, pH 9.2, for some plasmids pH 7.9 60ul
10/40dNTP mixture, final concentration of each l00umol/L 6ul
5mol/L betaine (final concentration 1.2mol/L), some plasmids with final concentration up to 2mol/L 156ul
H20 345ul
KlentaqLA (if target DNA is less than 2kb, just add lul) 3ul
Mixing
2. Take out 48ul, add 2ul primer as control.
3. Add l0ng/ul genomic DNA to the remaining solution and mix well.
4. Take 48ul from each tube.
5. Add 1ul of each upstream and downstream primer to each tube, or mix the upstream and downstream primers together and add 2ul. Do not worry about uneven mixing, because the convection during PCR amplification is sufficient to mix the primers.
6. When programming PCR amplification, the heating time for each cycle is about 5~10s, some PCR instruments may indicate 5~50s, and it may vary from one PCR instrument to another.
Each cycle is 50s at 92°C and 10 min at 63°C. Set the number of cycles as follows. 