Team:Washington/GENERAL PCR PROTOCOL

From 2013.igem.org

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<b>General Digestion Protocol in PCR Tubes</b>
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</header>
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<body>
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<p>
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2013 UW iGEM team uses Xba1 and Pst1 <a href = "https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1=%7B2DAF5F0E-59C4-4A5D-8809-6CEAD6BAE4B4%7D&enzyme2=%7B90EFE184-8442-45F0-8286-04CA22EEFFF2%7D/">combination</a>for most digestions
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<br>
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XbaI and SpeI together causes 50% inverted insertions and XbaI is less expensive than SpeI.
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<br>
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<br>
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<b>Digestion:</b>
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<br>
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10-42.5 uL DNA (200ng/uL = 2ug) Use the highest concentration (>50ng/ul) DNA available.*
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<br>
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DI water to 50 uL (33.5ul for single digest or 32.5 uL for double digest)
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<br>
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5uL of 10X buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) Use CutSmart with all ‘HF’ enzymes and XbaI.<br>
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0.5 uL BSA (If required. Not required for CutSmart. See above link) **<br>
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1uL enzyme 1 (kept on ice or freezer block)<br>
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For double digest add:<br>
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<u>1uL enzyme 2 (kept on ice)***</u><br>
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Total 50ul<br>
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(Enzymes can added last so they go into a 1X buffer concentration)<br>
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<u>Mix the reaction *well*</u> by pipetting up and down a few times (to mix the 50% glycerol).<br>
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<u>Incubate at 37C</u> for the required amount of time, usually 1 hr. Digests overnight work well.<br>
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<br>
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If double digest is required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications.
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<br><br>
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*1 unit of enzyme is defined as the amount needed to fully digest 1 ug of DNA in a 50 ul reaction in 1 hour. You can calculate how much enzyme and time is needed to do ~10-fold overdigestion.<br>
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**CutSmart contains BSA and is otherwise equivalent to Buffer 4 (except for DTT).<br>
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***Note that the enzymes are stored in 50% glycerol. The total RXN needs to be no more than 5% glycerol to get effective digestion. Therefore, the volume of enzyme you add should be no more than 10% of the total reaction volume. <br>
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****Thawing of buffer goes quickly when partially immersed in water. <br>
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<br>
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See also:<br>
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<a href = "http://bio.freelogy.org/wiki/Restriction_Digestion/">Restriction Digestion - FreeBio</a>
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<br><br><br>
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Diagnostic Digest: use 10-20 uL Total
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<br>
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<table border="1">
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<tr>
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<td>15 ul</td>
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<td>25 ul</td>
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</tr>
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<tr>
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<td>10-20 uL of DNA<br>
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2.5uL 10x Buffer<br>
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(0.5uL of BSA)<br>
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0.5 - 10 uL of Enz 1<br>
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0.5 - 10 uL of Enz 2<br>
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Fill to 25 uL with H20 <br>
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25 uL Total</td>
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<td>11 uL of DNA<br>
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1.5 uL of buffer 10x<br>
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(0.5 uL of BSA)<br>
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0.5 - 1.0 uL of Enz 1<br>
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0.5 - 1.0 uL of Enz 2<br>
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15 uL Total</td>
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</tr>
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</table>
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</p>
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</body>
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</html>

Revision as of 06:19, 7 September 2013

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General Digestion Protocol in PCR Tubes

2013 UW iGEM team uses Xba1 and Pst1 combinationfor most digestions
XbaI and SpeI together causes 50% inverted insertions and XbaI is less expensive than SpeI.

Digestion:
10-42.5 uL DNA (200ng/uL = 2ug) Use the highest concentration (>50ng/ul) DNA available.*
DI water to 50 uL (33.5ul for single digest or 32.5 uL for double digest)
5uL of 10X buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) Use CutSmart with all ‘HF’ enzymes and XbaI.
0.5 uL BSA (If required. Not required for CutSmart. See above link) **
1uL enzyme 1 (kept on ice or freezer block)
For double digest add:
1uL enzyme 2 (kept on ice)***
Total 50ul
(Enzymes can added last so they go into a 1X buffer concentration)
Mix the reaction *well* by pipetting up and down a few times (to mix the 50% glycerol).
Incubate at 37C for the required amount of time, usually 1 hr. Digests overnight work well.

If double digest is required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications.

*1 unit of enzyme is defined as the amount needed to fully digest 1 ug of DNA in a 50 ul reaction in 1 hour. You can calculate how much enzyme and time is needed to do ~10-fold overdigestion.
**CutSmart contains BSA and is otherwise equivalent to Buffer 4 (except for DTT).
***Note that the enzymes are stored in 50% glycerol. The total RXN needs to be no more than 5% glycerol to get effective digestion. Therefore, the volume of enzyme you add should be no more than 10% of the total reaction volume.
****Thawing of buffer goes quickly when partially immersed in water.

See also:
Restriction Digestion - FreeBio


Diagnostic Digest: use 10-20 uL Total

15 ul 25 ul
10-20 uL of DNA
2.5uL 10x Buffer
(0.5uL of BSA)
0.5 - 10 uL of Enz 1
0.5 - 10 uL of Enz 2
Fill to 25 uL with H20
25 uL Total		 11 uL of DNA
1.5 uL of buffer 10x
(0.5 uL of BSA)
0.5 - 1.0 uL of Enz 1
0.5 - 1.0 uL of Enz 2
15 uL Total

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