Team:USTC/Experiments
Protocols
Plasmid Extraction
Materials :
Buffer P1 (stored in 4 degree)
Buffer P2
Buffer p3
VisualLyse
Buffer DW1
Wash Solution dd
ddH2O
Prepation
1.Check out whether RNaseA has been added in Buffer P1.
2.Check out whether ethyl alcohol has been added in Wash Solution
3.Check out whether sediment exist in Buffer P2 and P2. Procedure
4.Absorb 1.5 to 5 mL bacteria solution in to EP tubes, centrifuge them at 8,000xg 2 mins and then discard the culture medium.
5.Add 250 ul Buffer P1 into sediment, and use spearhead to make bacteria suspended.
6.Add 250 ul Buffer P2, and overturn the EP tubes 5 to 10 times immediately and tenderly. Stand tubes about 2 to 4 mins.
7.Add 350 ul Buffer P3 and overturn the EP tubes 5 to 10 times again immediately and tenderly.
8.Centrifuge tubes at 12,000xg about 5 to 10 mins.
9.Pour the supernatant liquid into absorption columns ,centrifuge them at 8,000xg about 30 sec and then discard the liquid in the collecting pipe.
10.optional:Add 500 ul Buffer DW1 and centrifuge them at 9,000xg about 30 sec. Then discard the liquid in the collecting pipe.
11.Add 500 ul Wash Solution, centrifuge them at 9,000xg about 30 sec and discard the liquid in the collecting pipe.
12.Do the step 11 again.
13.Centrifuge the empty columns at 9,000xg about 1 min.
14.Put the columns in clean 1.5mL EP tubes, add 50 to 100 ul Elution Buffer on the absorption film, stand 10 min and centrifuge about 1 min at 9,000xg.
15.Keep the DNA solution for further work. The Protocol is based on SanPrep Column Type DNA Plasmid Extraction Kit.
PCR System Preparation and Conditions Setting
PCR system(set 50ul system as an example): The amount of substance of premier is written on the EP tubes. Calculating the volume of 1XTE required for 10uM primer solution is required before the system preparation.
1.Template DNA xul as required.
2.Forward Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
3.Reverse Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
4.5xTransStart FastPfu Fly Buffer 10 ul.
5.2.5 mM dNTPs 5 uM. The final concentration will be 0.25 mM.
6.TransStart FastPfu Fly DNA Polymerase 1 ul.
7.Double distilled water added to 50 ul. Reaction conditions
8.Calculate the Tm Value. If the complementary strip is smaller than 20 bp, Tm=4X(A+T)+2X(C+G), and the annealing temperature is Tm-5 degree centigrade. If not, use software to figure out the exact Tm value.(Also Tm value exist in report) PCR cycle:
9.1 cycle of 95 degree centigrade about 2 min for pre-degeneration;
10.30 to 35 cycles of 95 degree centigrade about 10 sec, Tm-5 degree centigrade about 20 sec and 72 degree centigrade 1kb per min;
11.1 cycle of 75 degree centigrade about 5 mins and stay the device in 4 degree centigrade.
The protocol is based on TransStart FastPfu Fly DNA Polymerase.
Materials
