Haynes Lab:Notebook/Engineering PC-TFs/2015/05/18: Difference between revisions
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==Summary== | ==Summary== | ||
* | * Restriction Digest/Gel Verification, Concentration Reading<br> | ||
'''Digest and Gel Verification''' | |||
<br> | |||
{| {{table}} border="1" cellspacing="5" | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="9" | | |||
<u><b>Expected:</b></u> | |||
<br> | |||
<u> V0120 [no cut sites]</u><br> | |||
3200 bp <br> <br> | |||
<u> pUC57 [KpnI]</u><br> | |||
Linearize: 2900 bp | |||
| rowspan="10" | [[Image:5-18-15.jpg|400px|]]<br> | |||
20 μL/lane; 1% agarose;<br> | |||
<u>Observed lengths:</u> <br> | |||
W1: Ladder <br> | |||
W2: KAH 021 w/ enzyme <br> | |||
W3: KAH 021 w/o enzyme <br> | |||
W4: KAH 022 w/ enzyme <br> | |||
W5: KAH 022 w/o enzyme <br> | |||
W6: KAH 023 w/ enzyme <br> | |||
W7: KAH 023 w/o enzyme <br> | |||
W8: KAH 024 w/ enzyme <br> | |||
W9: KAH 024 w/o enzyme <br> | |||
[http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA|| 5.0 μL 5.0 μL 8.0 μL 8.0 μL 5.0 μL 5.0 μL 5.0 μL 5.0 μL (From ladder 2 to ladder 9) | |||
|- | |||
| 10X buffer || 2.0 μL (for all) | |||
|- | |||
| KpnI || 1.0 μL (for even numbered wells only) | |||
|- | |||
| dH<sub>2</sub>O || 8.5 μL 8.5 μL 9.5 μL 9.5 μL 10.5 μL 10.5 μL | |||
|- | |||
| Total || 20 μL --> 37°C/ 15 min. | |||
|} | |||
<br> | |||
Note: This experiment is to verify what backbone the linker is in. There are two wells for each linker, one contains the restriction enzyme KpnI while the other does not. V0120 does not have a cut site for KpnI. If the two wells look the same, the linker is in V0120. If the two wells look different, the linker is in pUC57. As seen in the image, all the wells without the enzyme ran through the gel faster than all the wells with this enzyme. This indicates that it is a supercoiled plasmid as plasmids run faster than linear DNA. | |||
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Latest revision as of 00:58, 27 September 2017
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Summary
Digest and Gel Verification
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