Revision as of 14:54, 16 November 2012

PcTF Subcloning in E. coli

11/16/12

---Karmella 13:51, 16 November 2012 (EST): Here is my suggested procedure for trying the PcTF fusion cloning...

Assemblies: Shown as Part/cuts/purified fragment size

Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation
Note: KAH111-KAH151 are all cut and purified already

> Digest (Fermentas FD)

@@ Line 20: / Line 20: @@
 # KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
 # KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
+Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation<br>
+Note: KAH111-KAH151 are all cut and purified already
+> Digest (Fermentas FD)<br>
+# pT7CFE1-CHis, E/P
+{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
+|-valign="top"
+| <u>Reagent</u> || <u>Volume</u> || &nbsp;
+| rowspan="9" | [[Image:KAH_060311_gel1.tif|350px|Cloning digests 6/03/11]]<br>30 μL/lane, 1% agarose
+|-
+| DNA (plasmid) || 25.0 μL
+|-
+| 10x buffer || 3.0
+|-
+| EcoRI || 1.0
+|-
+| PstI || 1.0
+|-
+| dH<sub>2</sub>O || 0
+|-
+| &nbsp; || 30 μL --> 37°C/ ~30 min.
+|}