Current revision

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)

Plates Nomenclature

@@ Line 27: / Line 27: @@
 '''Digest (Fermentas FD)'''<br>
 # pT7CFE1-CHis, E/P
+# KAH109 E/P
+# KAH111 E/P
+# KAH112 E/P
+# KAH115 E/P
+# KAH148 E/P
+# KAH150 E/P
+# KAH151 E/P
 {| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
 |- valign="top"
 | <u>Reagent</u> || <u>Volume</u> || &nbsp;
-| rowspan="9" | <!--- [[Image:image.tif|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
+| rowspan="9" |   [[Image:PcTF_in-vitro_Expression.png‎|350px|Cloning digest ]]<br>30 μL/lane, 1% agarose
 |-
 | DNA (plasmid) || 25.0 μL
@@ Line 69: / Line 76: @@
 | 8. KAH151  (E/P) || --- || 1.9 || 19.701
 |}
@@ Line 89: / Line 95: @@
 | T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || 1.0
 |-
-| dH<sub>2</sub>O    || ---  || ---  || ---  || ---  || ---  || ---  || ---  || ---
+| dH<sub>2</sub>O    || 1.83  || 0.01  || 1.08  || 1.01  || 1.43  || 1.1  || 1.08  || 3.13
 |-
 | &nbsp;             || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL
 |}
+'''Plates Nomenclature
+# BD-116, (KAH109)
+# BD-117, (KAH111)
+# BD-118, (KAH112)
+# BD-119, (KAH115)
+# BD-120, (KAH148)
+# BD-121, (KAH150)
+# BD-122, (KAH151)
+# CTRL
 * Add total ligation reaction to 30 μL DH5α-Turbo cells
 * Follow routine "fast" transformation procedure
 * Plate onto 100 ug/mL Amp agar plates