Exeter/28 August 2013

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Latest revision as of 20:53, 2 October 2013

Exeter iGEM 2013 · Paint by Coli

NanoDrop

We collected NanoDrop data of previously miniprepped, defrosted DNA. The NanoDrop machine was zero-ed using the elution buffer from the gel extraction kit used.

ompC A - 20.1 ng/µl
ompC B - 9.3ng/µl
[http://parts.igem.org/Part:BBa_B0034?title=Part:BBa_B0034 B0034] - 26.7ng/µl
RFP - 198ng/µl

The concentration for ompC B was too low, so we continued on to the digestion with only ompC A. The concentration for RFP was too high, so we diluted it.

RFP (dilution) - 50.4ng/µl

Digestion

Part	ompC (Part A)	B0034 (Part B)	AMP Plasmid	RFP (Control)
DNA	12.4 ul	9.4 ul	10.0 ul	5.0 ul
Non-nuclease Water	3.6 ul	6.6 ul	5.5 ul	11.0 ul
NEB Buffer	2.5 ul	2.5 ul	2.5 ul	2.5 ul
BSA	0.5 ul	0.5 ul	0.5 ul	0.5 ul
EcoRI	0.5 ul	-	0.5 ul	0.5 ul
SpeI	0.5	-	-	-
XbaI	-	0.5 ul	-	-
PstI	-	0.5 ul	0.5 ul	0.5 ul
DpnI	-	-	0.5 ul	-

We pipetted the following volumes into four 0.6 ml tubes, keeping all enzymes and buffers on ice. We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.

The tubes were incubated at 25^oC for 30 minutes and then 80^oC for 20 minutes.

Ligation

The ligation was carried out using two methods: equal volumes and equal moles. To calculate the equimolar volumes, we used the following data:

Part	Part Length (bp)
AMP plasmid	2155
ompC (R0084)	108
RBS (B0034)	12
RFP	774

The ompC, B0034, AMP plasmid, and RFP were from the earlier digestions. We pippetted the following volumes into four 0.6ml tubes:

New Part	A (ompC + B0034, eq/vol)	B (RFP control, eq/vol)	C (ompC + B0034, equimolar)	C(RFP Control, equimolar)
Plasmid	2 ul	2 ul	2 ul	2 ul
ompC	2 ul	-	-	-
ompC (10x dilution)	-	-	1.0 ul	-
B0034	2 ul	-	-	-
B0034 (100x dilution)	-	-	0.8 ul	-
RFP	-	2 ul	-	-
RFP (10x diltion)	-	-	-	0.74 ul
T4 ligase buffer	1.0 ul	1.0 ul	1.0 ul	1.0 ul
T4 DNA ligase	0.5 ul	0.5 ul	0.5 ul	0.5 ul
Non-nuclease water	2.5 ul	4.5 ul	4.7 ul	5.8 ul

We flicked and centrifuged the tubes to ensure mixtures we homogeneous and collected at the bottom.

The tubes were incubated at 16^oC for 30 minutes and then 80^oC for 20 minutes.

Gel of Digestion

While carrying out the ligation, we ran a gel of our digested parts.

Take me back to the notebook.

Exeter iGEM 2013 · Paint by Coli

@@ Line 14: / Line 14: @@
 *RFP - 198ng/&micro;l
-The concentration for <i>ompF</i> B was too low, so we continued on to the digestion with only <i>ompF</i> A.
+The concentration for <i>ompC</i> B was too low, so we continued on to the digestion with only <i>ompC</i> A.
 The concentration for RFP was too high, so we diluted it.
@@ Line 24: / Line 24: @@
 {| class="wikitable"
 |-
-! Part !! <i>ompF</i> (Part A) !! B0034 (Part B) !! AMP Plasmid !! RFP (Control)
+! Part !! <i>ompC</i> (Part A) !! B0034 (Part B) !! AMP Plasmid !! RFP (Control)
 |-
 | DNA || 12.4 ul || 9.4 ul || 10.0 ul || 5.0 ul
@@ Line 60: / Line 60: @@
 | AMP plasmid || 2155
 |-
-| <i>ompF</i> (R0084) || 108
+| <i>ompC</i> (R0084) || 108
 |-
 | RBS (B0034) || 12
@@ Line 69: / Line 69: @@
-The <i>ompF</i>, B0034, AMP plasmid, and RFP were from the earlier digestions.
+The <i>ompC</i>, B0034, AMP plasmid, and RFP were from the earlier digestions.
 We pippetted the following volumes into four 0.6ml tubes:
@@ Line 75: / Line 75: @@
 {| class="wikitable"
 |-
-! New Part !! A (<i>ompF</i> + B0034, eq/vol) !! B (RFP control, eq/vol) !! C (<i>ompF</i> + B0034, equimolar) !! C(RFP Control, equimolar)
+! New Part !! A (<i>ompC</i> + B0034, eq/vol) !! B (RFP control, eq/vol) !! C (<i>ompC</i> + B0034, equimolar) !! C(RFP Control, equimolar)
 |-
 | Plasmid || 2 ul || 2 ul || 2 ul || 2 ul
 |-
-| <i>ompF</i> || 2 ul || - || - || -
+| <i>ompC</i> || 2 ul || - || - || -
 |-
-| <i>ompF</i> (10x dilution) || - || - || 1.0 ul || -
+| <i>ompC</i> (10x dilution) || - || - || 1.0 ul || -
 |-
 | B0034 || 2 ul || - || - || -