Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
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<ol start="2"> | <ol start="2"> | ||
<li>Confirm the size of the digested product by gel electrophoresis.</li> | <li>Confirm the size of the digested product by gel electrophoresis.</li> | ||
− | + | </br> | |
<li>Gel purification of the target size.</li> | <li>Gel purification of the target size.</li> | ||
</ol> | </ol> | ||
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<ol start="3"> | <ol start="3"> | ||
<li>Confirm the size of the digested product by gel electrophoresis.</li> | <li>Confirm the size of the digested product by gel electrophoresis.</li> | ||
+ | </br> | ||
<li>Gel purification of the target size.</li> | <li>Gel purification of the target size.</li> | ||
+ | </br> | ||
<li>Ligation</li> | <li>Ligation</li> | ||
</ol> | </ol> | ||
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<li>Gel Extraction</li> | <li>Gel Extraction</li> | ||
<ol type="a"> | <ol type="a"> | ||
− | <li>Excised the DNA fragment from the agarose gel.</li> | + | <li>Excised the DNA fragment from the agarose gel.</li></br> |
<li>Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge | <li>Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge | ||
− | tube.</li> | + | tube.</li></br> |
− | <li>Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex.</li> | + | <li>Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex.</li></br> |
<li>Incubate at 55~60℃ for 10 minutes (or until the gel slice has | <li>Incubate at 55~60℃ for 10 minutes (or until the gel slice has | ||
− | completely dissolved).</li> | + | completely dissolved).</li> </br> |
− | <li>During the incubation, mixed by vortexing the tube every 2~3 minutes.</li> | + | <li>During the incubation, mixed by vortexing the tube every 2~3 minutes.</li></br> |
<li>Cooled the dissolved sample mixture to the room temperature.</li> | <li>Cooled the dissolved sample mixture to the room temperature.</li> | ||
</ol> | </ol> | ||
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<ol start="2"> | <ol start="2"> | ||
<li>Placed a PG Column in a Collection Tube. Apply the supernatant to the PG | <li>Placed a PG Column in a Collection Tube. Apply the supernatant to the PG | ||
− | Column by decanting or pipetting.</li> | + | Column by decanting or pipetting.</li></br> |
− | <li>Centrifuged at 16,000 xg for 30 seconds.</li> | + | <li>Centrifuged at 16,000 xg for 30 seconds.</li></br> |
− | + | ||
<li>Discarded the flow-through and place the PG Column back into the same | <li>Discarded the flow-through and place the PG Column back into the same | ||
− | collection tube.</li> | + | collection tube.</li></br> |
</ol> | </ol> | ||
</p> | </p> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
<ol start="5"> | <ol start="5"> | ||
− | <li>Added 400 μl of the Buffer W1 into the PG Column.</li> | + | <li>Added 400 μl of the Buffer W1 into the PG Column.</li></br> |
− | <li>Centrifuged at 16,000 xg for 30 seconds.</li> | + | <li>Centrifuged at 16,000 xg for 30 seconds.</li></br> |
<li>Discarded the flow-through and place the PG Column back into the same | <li>Discarded the flow-through and place the PG Column back into the same | ||
− | collection tube.</li> | + | collection tube.</li></br> |
− | <li>Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.</li> | + | <li>Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.</li></br> |
− | <li>Centrifuged at 16,000 xg for 30 seconds.</li> | + | <li>Centrifuged at 16,000 xg for 30 seconds.</li></br> |
<li>Discarded the flow-through and place the PG Column back into the same | <li>Discarded the flow-through and place the PG Column back into the same | ||
− | collection tube.</li> | + | collection tube.</li></br> |
<li>Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer | <li>Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer | ||
W2.</li> | W2.</li> | ||
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<li>To elute the DNA, placed the PG Column in a clean 1.5 ml | <li>To elute the DNA, placed the PG Column in a clean 1.5 ml | ||
microcentrifuge | microcentrifuge | ||
− | tube.</li> | + | tube.</li></br> |
<li>Added 50 μl of the H2O (pH is between 7.0 and 8.5) to the center of | <li>Added 50 μl of the H2O (pH is between 7.0 and 8.5) to the center of | ||
− | + | each PG | |
− | + | Column, let it stand for at least 2 minutes, and centrifuge at 16,000 xg | |
− | + | for 2 | |
+ | min.</li> | ||
</ol> | </ol> | ||
</p> | </p> | ||
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<li>Streak out wild type E. coli on a plate (LB plate without antibiotics) | <li>Streak out wild type E. coli on a plate (LB plate without antibiotics) | ||
overnight | overnight | ||
− | and pick one colony into 3 ml of media (LB) and grow overnight.</li> | + | and pick one colony into 3 ml of media (LB) and grow overnight.</li></br> |
<li>Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at | <li>Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at | ||
37 | 37 | ||
− | ℃.</li> | + | ℃.</li></br> |
− | <li>When the OD600 nm up to 0.35, put the cells on ice immediately.</li> | + | <li>When the OD600 nm up to 0.35, put the cells on ice immediately.</li></br> |
− | <li>Spin the cells at 4℃for 10 minutes at 4000 rpm.</li> | + | <li>Spin the cells at 4℃for 10 minutes at 4000 rpm.</li></br> |
<li>Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer | <li>Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer | ||
− | 1(TFB1).</li> | + | 1(TFB1).</li></br> |
− | <li>Leave nicely suspended bugs on ice for 10 minutes.</li> | + | <li>Leave nicely suspended bugs on ice for 10 minutes.</li></br> |
− | <li>Spin the cells at 4℃ for 10 min. at 4000 rpm.</li> | + | <li>Spin the cells at 4℃ for 10 min. at 4000 rpm.</li></br> |
− | <li>Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).</li> | + | <li>Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).</li></br> |
− | <li>Leave on immediately on ice for 30 minutes.</li> | + | <li>Leave on immediately on ice for 30 minutes.</li></br> |
<li>Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with | <li>Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with | ||
− | liquid nitrogen.</li> | + | liquid nitrogen.</li></br> |
− | <li>Store the frozen cells in the -80℃ freezer.</li> | + | <li>Store the frozen cells in the -80℃ freezer.</li></br> |
</ol> | </ol> | ||
</p> | </p> | ||
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<ol> | <ol> | ||
<li>Transfer 1.4 ml of well-grown bacterial culture to | <li>Transfer 1.4 ml of well-grown bacterial culture to | ||
− | a centrifuge tube.</li> | + | a centrifuge tube.</li></br> |
<li>Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and | <li>Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and | ||
− | discard the supernatant completely.</li> | + | discard the supernatant completely.</li></br> |
<li>Add 200 µl of FAPD1 Buffer (RNaseA added) to the cell pellet and resuspend | <li>Add 200 µl of FAPD1 Buffer (RNaseA added) to the cell pellet and resuspend | ||
− | the cells completely by pipetting.</li> | + | the cells completely by pipetting.</li></br> |
<ul> | <ul> | ||
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<li>No cell pellet should be visible after resuspension of the cells.</li> | <li>No cell pellet should be visible after resuspension of the cells.</li> | ||
− | </ul> | + | </ul></br> |
<li>Add 200 µl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times. | <li>Add 200 µl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times. | ||
Incubate | Incubate | ||
− | the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells.</li> | + | the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells.</li></br> |
<ul> | <ul> | ||
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<li>Make sure the tube transfer to clarify from turbid.</li> | <li>Make sure the tube transfer to clarify from turbid.</li> | ||
<li>Do not proceed with the incubation over 5 minutes.</li> | <li>Do not proceed with the incubation over 5 minutes.</li> | ||
− | </ul> | + | </ul></br> |
<li>Add 300 µl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to | <li>Add 300 µl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to | ||
− | neutralize the lysate.</li> | + | neutralize the lysate.</li></br> |
<ul> | <ul> | ||
<li>Invert immediately after adding FAPD3 Buffer will avoid asymmetric | <li>Invert immediately after adding FAPD3 Buffer will avoid asymmetric | ||
precipitation.</li> | precipitation.</li> | ||
− | </ul> | + | </ul></br> |
<li>Centrifuge at 16,000 xg for 3 minutess. to clarify the lysate. During | <li>Centrifuge at 16,000 xg for 3 minutess. to clarify the lysate. During | ||
− | centrifugation, place a FAPD Column in a Collection Tube.</li> | + | centrifugation, place a FAPD Column in a Collection Tube.</li></br> |
<li>Transfer the supernatant carefully to the FAPD Column and centrifuge at | <li>Transfer the supernatant carefully to the FAPD Column and centrifuge at | ||
16,000 xg for 1 minute. Discard the flow-through and place the column back | 16,000 xg for 1 minute. Discard the flow-through and place the column back | ||
to | to | ||
− | the Collection Tube.</li> | + | the Collection Tube.</li></br> |
<ul> | <ul> | ||
<li>Do not transfer any white pellet into the column.</li> | <li>Do not transfer any white pellet into the column.</li> | ||
− | </ul> | + | </ul></br> |
<li>Add 400 µl of W1 Buffer to the FAPD Column and centrifuge at 16,000 xg for | <li>Add 400 µl of W1 Buffer to the FAPD Column and centrifuge at 16,000 xg for | ||
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minute. Discard the flow-through and place the column back to the | minute. Discard the flow-through and place the column back to the | ||
Collection | Collection | ||
− | Tube.</li> | + | Tube.</li></br> |
<li>Add 600 µl of Wash Buffer to the FAPD Column and centrifuge at 16,000 xg | <li>Add 600 µl of Wash Buffer to the FAPD Column and centrifuge at 16,000 xg | ||
for | for | ||
1 minute. Discard the flow-through and place the column back to the | 1 minute. Discard the flow-through and place the column back to the | ||
Collection | Collection | ||
− | Tube.</li> | + | Tube.</li></br> |
<ul> | <ul> | ||
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when | when | ||
first use.</li> | first use.</li> | ||
− | </ul> | + | </ul></br> |
<li>Centrifuge at 16,000 xg for an additional 3 minutes to dry the FAPD | <li>Centrifuge at 16,000 xg for an additional 3 minutes to dry the FAPD | ||
− | Column.</li> | + | Column.</li></br> |
<ul> | <ul> | ||
Line 487: | Line 489: | ||
this | this | ||
step.</li> | step.</li> | ||
− | </ul> | + | </ul></br> |
− | <li>Place the FAPD Column to a new 1.5 ml microcentrifuge tube.</li> | + | <li>Place the FAPD Column to a new 1.5 ml microcentrifuge tube.</li></br> |
<li>Add 30 µl of Elution Buffer or ddH2O to the membrane center of the FAPD | <li>Add 30 µl of Elution Buffer or ddH2O to the membrane center of the FAPD | ||
− | Column. Stand the column for 3 minute.</li> | + | Column. Stand the column for 3 minute.</li></br> |
<ul> | <ul> | ||
<li>Important step! For effective elution, make sure that the elution | <li>Important step! For effective elution, make sure that the elution | ||
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<li>Do not elute the DNA using less than suggested volume (50 µl). It will | <li>Do not elute the DNA using less than suggested volume (50 µl). It will | ||
lower the final yield.</li> | lower the final yield.</li> | ||
− | </ul> | + | </ul></br> |
<li>Centrifuge at 16,000 xg for 3 minute to elute plasmid DNA and store the DNA | <li>Centrifuge at 16,000 xg for 3 minute to elute plasmid DNA and store the DNA | ||
− | + | at -20 ℃.</li> | |
</ol> | </ol> | ||
</p> | </p> |
Revision as of 12:17, 29 September 2018