Exeter/28 August 2013

From 2013.igem.org

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 25oC for 30 minutes and then 80oC 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 16oC for 30 minutes and then 80oC 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
Retrieved from "http://2013.igem.org/Exeter/28_August_2013"