Exeter/28 August 2013

From 2013.igem.org

(Difference between revisions)
Line 37: Line 37:
|-
|-
| Dpn1 || - || - || 0.5 ul || -
| Dpn1 || - || - || 0.5 ul || -
 +
|}
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 +
 +
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 25C for 30 minutes and then 80C 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:
 +
 +
{| class="wikitable"
 +
|-
 +
! Part !! Part Length (bp)
 +
|-
 +
| AMP plasmid || 2155
 +
|-
 +
| OmpF (R0084) || 108
 +
|-
 +
| RBS (B0034) || 12
 +
|-
 +
| RFP || 774
 +
|}
 +
 +
 +
 +
The Ompf, B0034, AMP plasmid, and RFP were from the earlier digestions.
 +
We pippetted the following volumes into four 0.6ml tubes:
 +
 +
 +
{| class="wikitable"
 +
|-
 +
! New Part !! A (OmpF + B0034, eq/vol) !! B (RFP control, eq/vol) !! C (OmpF + B0034, equimolar) !! C(RFP Control, equimolar)
 +
|-
 +
| Plasmid || 2 ul || 2 ul || 2 ul || 2 ul
 +
|-
 +
| Ompf || 2 ul || - || - || -
 +
|-
 +
| Ompf (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 ||
|}
|}

Revision as of 14:05, 28 August 2013

NanoDrop data of the miniprepped, defrosted DNA:

The NanoDrop machine was zero-ed using the elution buffer from the gel extraction kit used.

  • OmpF A - 20.1 ng/ul
  • OmpF B - 9.3ng/ul
  • B0034 - 26.7ng/ul
  • RFP - 198ng/ul

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

  • RFP (dilution) - 50.4ng/ul

Digestion

Part OmpF (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
EcoR1 0.5 ul - 0.5 ul 0.5 ul
Spe1 0.5 - - -
Xbal1 - 0.5 ul - -
Pst1 - 0.5 ul 0.5 ul 0.5 ul
Dpn1 - - 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 25C for 30 minutes and then 80C 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
OmpF (R0084) 108
RBS (B0034) 12
RFP 774


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


New Part A (OmpF + B0034, eq/vol) B (RFP control, eq/vol) C (OmpF + B0034, equimolar) C(RFP Control, equimolar)
Plasmid 2 ul 2 ul 2 ul 2 ul
Ompf 2 ul - - -
Ompf (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
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