Revision as of 16:29, 26 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)

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 53: / Line 53: @@
 | <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
 |-
-| 1. pT7CFE1-CHis (E/P) || --- || --- || ---
+| 1. pT7CFE1-CHis (E/P) || --- || 1.829 || 57.344
+|-
+| 2. KAH109  (E/P) || --- || 2.354 || 36.072
+|-
+| 3. KAH111  (E/P) || --- || 2.238 || 15.003
+|-
+| 4. KAH112  (E/P) || --- || 1.862 || 22.857
+|-
+| 5. KAH115  (E/P) || --- || 2.096 || 18.312
+|-
+| 6. KAH148  (E/P) || --- || 2.389 || 23.735
+|-
+| 7. KAH150  (E/P) || --- || 1.99 || 19.822
+|-
+| 8. KAH151  (E/P) || --- || 1.9 || 19.701
 |}
@@ Line 65: / Line 79: @@
 | &nbsp;             || 1    || 2    || 3    || 4    || 5    || 6    || 7    || 8
 |-
-| Insert DNA         || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
+| Insert Name      || KAH109  || KAH111  || KAH112  || KAH115  || KAH148  || KAH150  || KAH151  || N/A
+|-
+| Insert DNA         || 1.3  || 3.12 || 2.05  || 2.12  || 1.7 || 2.03  || 2.05  || 0
 |-
-| Vector DNA         || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
+| Vector DNA         || 0.87  || 0.87  || 0.87  || 0.87  || 0.87  || 0.87  || 0.87  || 0.87
 |-
 | 2x lgn buf (Roche) || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0  || 5.0