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Team:ESBS-Strasbourg/Purification of PCR product
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(New page: '''Purification of classic PCR product''' <br> 1)Remarks: <br> In this case we obtain linear DNA which could be purified after a Gel migration or directly after the PCR. <br> {| class="...) |
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| - | '''Purification of classic PCR product''' <br> | + | '''I: Purification of classic PCR product''' <br> |
| - | + | a) Remarks <br> | |
In this case we obtain linear DNA which could be purified after a Gel migration or directly after the PCR. <br> | In this case we obtain linear DNA which could be purified after a Gel migration or directly after the PCR. <br> | ||
| Line 16: | Line 16: | ||
| - | + | b) Protocol <br> | |
See the Quiagen kit instruction for both types of purification. <br> | See the Quiagen kit instruction for both types of purification. <br> | ||
| - | '''Purification of mutagenesis PCR product''' | + | '''II: Purification of mutagenesis PCR product''' |
| - | + | a) Remarks <br> | |
Here we get the product in a plasmid form. It’s more difficult to purify the new plasmid (which own the mutation) from the original plasmid. There are several possibilities: <br> | Here we get the product in a plasmid form. It’s more difficult to purify the new plasmid (which own the mutation) from the original plasmid. There are several possibilities: <br> | ||
-Use of the Dpn1 enzyme, which recognize the methyl Dna, in other words the original plasmid would be degraded. In this case, just add Dpn1 in the PCR product, and after the degradation the solution can be directly employ for transformation <br> | -Use of the Dpn1 enzyme, which recognize the methyl Dna, in other words the original plasmid would be degraded. In this case, just add Dpn1 in the PCR product, and after the degradation the solution can be directly employ for transformation <br> | ||
-Purification after gel migration: difficult it will be very hard to distinguish plasmids which own the mutation and the other ones. One possibility could be to use Agarose Low melting point, or to decrease the agarose concentration (0,5 or 0,3). Then be careful cause the gel would be more difficult to handle (fragile) <br> | -Purification after gel migration: difficult it will be very hard to distinguish plasmids which own the mutation and the other ones. One possibility could be to use Agarose Low melting point, or to decrease the agarose concentration (0,5 or 0,3). Then be careful cause the gel would be more difficult to handle (fragile) <br> | ||
| - | see [[Agarose gel preparation]] | + | see [[Team:ESBS-Strasbourg/Agarose gel preparation|Agarose gel preparation]] |
| - | + | b) Protocol <br> | |
| - | -see [[Restriction digestion]] part for a Dpn1 digestion <br> | + | -see [[Team:ESBS-Strasbourg/Restriction digestion|Restriction digestion]] part for a Dpn1 digestion <br> |
-Follow the Quigen kit instruction for traditionnal purification <br> | -Follow the Quigen kit instruction for traditionnal purification <br> | ||
Take max. 400mg of gel, no more | Take max. 400mg of gel, no more | ||
| - | + | [[Team:ESBS-Strasbourg/Protocols|''Protocols'']] | |
| - | + | ||
| - | + | ||
Latest revision as of 13:48, 14 July 2008
I: Purification of classic PCR product
a) Remarks
In this case we obtain linear DNA which could be purified after a Gel migration or directly after the PCR.
| Purification's yield | |
| Directly after PCR | ~90% |
| After gel migration | ~75% |
b) Protocol
See the Quiagen kit instruction for both types of purification.
II: Purification of mutagenesis PCR product
a) Remarks
Here we get the product in a plasmid form. It’s more difficult to purify the new plasmid (which own the mutation) from the original plasmid. There are several possibilities:
-Use of the Dpn1 enzyme, which recognize the methyl Dna, in other words the original plasmid would be degraded. In this case, just add Dpn1 in the PCR product, and after the degradation the solution can be directly employ for transformation
-Purification after gel migration: difficult it will be very hard to distinguish plasmids which own the mutation and the other ones. One possibility could be to use Agarose Low melting point, or to decrease the agarose concentration (0,5 or 0,3). Then be careful cause the gel would be more difficult to handle (fragile)
see Agarose gel preparation
b) Protocol
-see Restriction digestion part for a Dpn1 digestion
-Follow the Quigen kit instruction for traditionnal purification
Take max. 400mg of gel, no more
