Editing PLenti-IRES-TFR-EGFP

CloneID: 252 

Date: 2011-6-24to 2011-6-28<br>

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'''PCR, DIGESTION, GEL&nbsp;ELECTROPHORESIS:'''<br> 

PCR1) 1ul F1, 1ul&nbsp;P8, 2ul primer mix, 5ul 5x buffer, 0.5ul 50x dNTP, 14.5ul H2O, 1ul pfu enzyme<br>

PCR2) 1ul F2, 1ulR, 1ul EGFP template, 5ul 5x buffer, 0.5ul dNTP, 15.5ul H2O, 1ul pfu enzyme<br>

PCR3) 5 ul PCR1, 5ul PCR2, 1ul F1, 1ul&nbsp;R, 8ul 5x buffer, 1ul dNTP, 2ul pfu enzyme, 27ul H2O<br>

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Used MluI+XhoI enzymes to digest, 100% in buffer 3 

1)&nbsp;pLentiplus vector:&nbsp;5ul pLentiplus, 0.4ul Xho1, 0.4ul Mlu1, 1.5ul buffer 3, 7.7ul H2O<br>

2) PCR3 product:&nbsp;12.7ul purified DNA, 0.4ul Xho1, 0.4ul Mlu1, 1.5ul buffer 3 (75ºC for ~15 min after digestion complete)<br>

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Ran agarose gel for pLentiplus vector and PCR3 product<br>

(Image to be uploaded later)<br>

Lanes: 

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'''COLONY&nbsp;SCREENING:'''<br>

PCR:<br>

11 colonies -<br>

each contains:&nbsp;1ul template (H2O+colony), 1ul A3, 1ul B12, 2.5ul 10x buffer, 0.8ul Taq enzyme, 0.2ul dNTP, 18.5ul H2O<br>


[[Image:PLenti-IRES-TFR-EGFP-ColonyScreening.jpg]] <br>

Lanes:<br>

Marker<br>

1: Marker

2: colony 1<br>

3: colony 2<br>

4: colony 3<br>

5: colony 4<br>

6: colony 5<br>

7: colony 6<br>

8: colony 7<br>

9: colony 8<br>

10: colony 9<br>

11: colony 10<br>

12: colony 11<br>

Took DNA from colonies 5-8 and 10-11

(pg. 001143)<br>

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[[Category:Cloning|Cloning]]