Revision as of 15:01, 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)

> Measure concentration(s)

Sample	OD260	260/280	ng/μL
1. pT7CFE1-CHis (E/P)	---	---	---

> Ligations (trouble shooting suggestions)

Note: Use 50 ng of vector this time (instead of 20)
Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)

@@ Line 55: / Line 55: @@
 |-
 | 1. pT7CFE1-CHis (E/P) || --- || --- || ---
+|}
+> Ligations (trouble shooting suggestions)
+* Note: Use 50 ng of vector this time (instead of 20)
+* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
+{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
+| &nbsp;             || 1    || 2    ||
+|-
+| Insert DNA         || 3.5  || ---  ||
+|-
+| Vector DNA         || 0.5  || 0.5  ||
+|-
+| 2x lgn buf (Roche) || 5.0  || 5.0  ||
+|-
+| T4 ligase (NEB)    || 1.0  || 1.0  ||
+|-
+| dH<sub>2</sub>O    || ---  || 3.5  ||
+|-
+| &nbsp;             || 10 μL || 10 μL ||
 |}