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| − | <br>
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| − | <p id="first" class="scrollspy label label-pink">Protocols</p>
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| − | <br>
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| − | <br>
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| − | <p>Plasmid Extraction</p>
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| − | <p>Materials
:</p>
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| − | <p>Buffer P1 (stored in 4 degree)
</p>
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| − | <p>Buffer P2
</p>
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| − | <p>Buffer p3
</p>
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| − | <p>VisualLyse</p>
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| − | <p>
Buffer DW1
</p>
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| − | <p>Wash Solution
dd </p>
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| − | <p>ddH<sub>2</sub>O</p>
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| − | <p>
Prepation
</p>
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| − | <p>1.Check out whether RNaseA has been added in Buffer P1.
</p>
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| − | <p>2.Check out whether ethyl alcohol has been added in Wash Solution
</p>
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| − | <p>3.Check out whether sediment exist in Buffer P2 and P2.
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| − | <p>Procedure
</p>
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| − | <p>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.</p>
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| − | <p>5.Add 250 ul Buffer P1 into sediment, and use spearhead to make bacteria suspended.
</p>
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| − | <p>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.
</p>
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| − | <p>7.Add 350 ul Buffer P3 and overturn the EP tubes 5 to 10 times again immediately and tenderly.
</p>
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| − | <p>8.Centrifuge tubes at 12,000xg about 5 to 10 mins.
</p>
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| − | <p>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.
</p>
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| − | <p>10.optional:Add 500 ul Buffer DW1 and centrifuge them at 9,000xg about 30 sec. Then discard the liquid in the collecting pipe.
</p>
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| − | <p>11.Add 500 ul Wash Solution, centrifuge them at 9,000xg about 30 sec and discard the liquid in the collecting pipe.</p>
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| − | <p>
12.Do the step 11 again.
</p>
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| − | <p>13.Centrifuge the empty columns at 9,000xg about 1 min.</p>
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| − | <p>
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.
</p>
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| − | <p>15.Keep the DNA solution for further work.
The Protocol is based on SanPrep Column Type DNA Plasmid Extraction Kit.</p>
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| | | | |
| − | <br>
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| − | <br>
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| − | <p>PCR System Preparation and Conditions Setting</p>
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| − | <p>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.</p>
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| − | <p>
1.Template DNA xul as required.
</p>
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| − | <p>2.Forward Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
</p>
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| − | <p>3.Reverse Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
</p>
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| − | <p>4.5xTransStart FastPfu Fly Buffer 10 ul.
</p>
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| − | <p>5.2.5 mM dNTPs 5 uM. The final concentration will be 0.25 mM.
</p>
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| − | <p>6.TransStart FastPfu Fly DNA Polymerase 1 ul.
</p>
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| − | <p>7.Double distilled water added to 50 ul.
Reaction conditions
</p>
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| − | <p>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:</p>
| |
| − | <p>
9.1 cycle of 95 degree centigrade about 2 min for pre-degeneration;
</p>
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| − | <p>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;
</p>
| |
| − | <p>11.1 cycle of 75 degree centigrade about 5 mins and stay the device in 4 degree centigrade.
</p>
| |
| − | <p>The protocol is based on TransStart FastPfu Fly DNA Polymerase.</p>
| |
| − |
| |
| − | <br>
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| − | <br>
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| − | <p>PCR of Bacteria System Preparation and Conditions Setting</p>
| |
| − | <p>PCR system(set 50ul system as an example):
</p>
| |
| − | <p>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.
</p>
| |
| − | <p>1.Template(bacteria fluid) about 1 ul.
</p>
| |
| − | <p>2.Forward Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
</p>
| |
| − | <p>3.Reverse Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.</p>
| |
| − | <p>
4.5xTransStart FastPfu Fly Buffer 10 ul.
</p>
| |
| − | <p>5.2.5 mM dNTPs 5 uM. The final concentration will be 0.25 mM.
</p>
| |
| − | <p>6.TransStart FastPfu Fly DNA Polymerase 1 ul.
</p>
| |
| − | <p>7.Double distilled water added to 50 ul.
Reaction conditions
.</p>
| |
| − | <p>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:</p>
| |
| − | <p>
9.1 cycle of 95 degree centigrade about 2 min for pre-degeneration;
</p>
| |
| − | <p>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;</p>
| |
| − | <p>
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.</p>
| |
| − | <p>PCR System Preparation and Conditions Setting
PCR system(set 50ul system as an example): </p>
| |
| − | <p>
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.
</p>
| |
| − | <p>1.Template DNA xul as required.
</p>
| |
| − | <p>2.Forward Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.</p>
| |
| − | <p>
3.Reverse Primer(10 uM) about 1 to 2 ul. The final concentration will be 0.2 to 0.4 uM.
</p>
| |
| − | <p>4.5xTransStart FastPfu Fly Buffer 10 ul.</p>
| |
| − | <p>
5.2.5 mM dNTPs 5 uM. The final concentration will be 0.25 mM.
</p>
| |
| − | <p>6.TransStart FastPfu Fly DNA Polymerase 1 ul.
</p>
| |
| − | <p>7.Double distilled water added to 50 ul.
Reaction conditions
</p>
| |
| − | <p>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:</p>
| |
| − | <p>
9.1 cycle of 95 degree centigrade about 2 min for pre-degeneration;
</p>
| |
| − | <p>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;
</p>
| |
| − | <p>11.1 cycle of 75 degree centigrade about 5 mins and stay the device in 4 degree centigrade.
</p>
| |
| − | <p>The protocol is based on TransStart FastPfu Fly DNA Polymerase.</p>
| |
| − |
| |
| − | <br>
| |
| − | <br>
| |
| − | <p>PCR Production Extraction</p>
| |
| − | <p>Prepation</p>
| |
| − |
<p>1.Check out whether ethyl alcohol has been added into Wash Solution.
</p>
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| − | <p>2.Check out whether sediment exist in Buffer B3.</p>
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| − |
<p>3.Check out whether isopropanol has been added into Buffer B3.</p>
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| − |
<p>Procedure</p>
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| − |
<p>4.Add Buffer B3 3 times volume of PCR system solution and incorporated thoroughly.
</p>
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| − | <p>5.Centrifuge them at 8,000xg about 30 sec and drain the liquid in the collecting pipe.</p>
| |
| − |
<p>6.Add 500 ul Wash Solution at 9,000xg about 30 sec and drain the liquid in the collecting pipe.</p>
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| − |
<p>7.Do The step 3 again.
</p>
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| − | <p>8.Centrifuge the empty pipes at 9,000Xg about 1 min.
</p>
| |
| − | <p>9.Put the absorption column in some clean 1.5 mL EP tubes, add 15 to 40 ul Elution Buffer, stand for 1 min and centrifuge them at 9,000xg about 1min.</p>
| |
| − |
<p>10.Keep the DNA solution.</p>
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| | | | |
| | </div> | | </div> |
