Team:Alberta/Notebook/Assembly Method

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Revision as of 17:31, 19 July 2010

10-05-2010

PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)

Recipe:

1μL p1003 (approx. 1ng)

2.5μL prA_p1003+

2.5μL prB'_p1003-

5μL 10X PCR buffer

1μL 10uM dNTPs

2μL 50uM MgCl₂

0.5μL Taq polymerase

35.5μL MilliQ H₂O

</br>

Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-.

Program:

5 min-94^oC
45 sec-94^oC
1 min-60^oC
1 min-72^oC
Repeat 2 through 4 35 times
5 min-72^oC

Notebook	Building Parts	Testing Parts	Assembly Method	Competent Cells	Plates	Kit Manual	Software

@@ Line 1: / Line 1: @@
+==10-05-2010==
+<p>PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends. (Fragments formed called KanR A/B'-Bsa and KanR B/A'-Bsa respectively)</p>
+<p>Recipe:
+:1&mu;L p1003 (approx. 1ng)
+:2.5&mu;L prA_p1003+
+:2.5&mu;L prB'_p1003-
+:5&mu;L 10X PCR buffer
+:1&mu;L 10uM dNTPs
+:2&mu;L 50uM MgCl<sub>2</sub>
+:0.5&mu;L Taq polymerase
+:35.5&mu;L MilliQ H<sub>2</sub>O</p>
+</br>
+<p>Same recipe for KanR B/A'-Bsa except primers are prB_p1003+ and prA'_p1003-. </p>
+<p>Program:
+# 5 min-94<sup>o</sup>C
+# 45 sec-94<sup>o</sup>C
+# 1 min-60<sup>o</sup>C
+# 1 min-72<sup>o</sup>C
+# Repeat 2 through 4 35 times
+# 5 min-72<sup>o</sup>C</p>
+<!--Image of gel performed that day. 5&mu;L of each PCR reaction, 1&mu;L of 10X loading dye and 4&mu;L MilliQ water in a 1% agarose gel -->
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