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Week 12
08/17 - 08/21
Gibson Assembly
- Gel Migration of the PCR products (Cluster 2 and BBa_808014)
- PCR Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.
- Gel Migration on 0,8% Agarose Gel
- PCR Amplification of BBa_316003 using Takara Ex Taq Polymerase.
- Gel Migration using 0,8% Agarose Gel
- Spe I and Pst I Restriction Digest of the recieved plasmids containing BBa_K936011, BBa_K13932932, K936023.
- Gel Purification of the digested plasmids K936011 and K1392932 using the QIAgen Gel extraction kit protocol.
NB -Esterase assay
- Liquid culture of the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in DH5α
- MiniPrep of BBa_808030 in the plasmid pDG011 in BAP 1
- XbaI and PstI digestion of BBa_808030 and pDG011 (with the phosphatase step)
Gel Purification
Tubes components:
Tubes |
Ladder |
1st transformation |
Components |
GeneRuler 1kb DNA Ladder
(6x DNA loading dye)
3µL |
Buffer
4µL
+
DNA
20µL |
- Ligation of BBa_808030 and pDG011
- Transformation in Chemically Competent DH5-⍺ cells
- Transformation in DH5-ɑ of the biobrick K808030 in pDG011
- Transformation in DH5α cells of BBa_808030 insert and pDG011 vector
- SpeI and Pst I Enzymatic Digest of BBa_K808030 with dephosphorylation.
- Gel migration
- Gel extraction of the PCR product of K808030 and pDG011.
- Ligation of pDG011 and K808030.
The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.
- Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.
- Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000)
|
DNA Concentration (ng/µl) |
OD(260nm) |
OD(280nm) |
OD(260nm)/OD(280nm) |
BBa_J61047 |
0,5 |
0,011 |
0,004 |
2,57 |
- Preparation of YPD specific media on specific media (1% yeast extract, 2% Dextrose, 2% Peptone, 2% agar).
- Gel Migration of BBa_J61047.
- NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme.
- Gel Migration on a 0,8% Agarose Gel and the digestion hasn’t work.
- XbaI and SpeI digestion of BBa_J61047.
- tube 1 « Miniprep XbaI/SpeI » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.
- tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour
- tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
- tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
- tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
- tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
- MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB
- PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.
- Gel Migration on a 0.8% agarose gel (See figure 01)
- Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.
- PCR Amplification using the Phusion Polymerase of pRS415.
NB-Esterase Assay:
|
Concentration (ng/µL) |
OD(230nm) |
OD(260nm) |
OD(280nm) |
OD(260nm)/OD(230nm) |
OD(260nm)/OD(280nm) |
Cluster_4_start |
30 |
0,025 |
0,575 |
0,375 |
1,50 |
- |
/936011 |
55 |
0,4 |
1,075 |
0,575 |
1,88 |
2,65 |
936011/936023 |
7,5 |
0,6 |
0,125 |
0,05 |
2,41 |
0,21 |
936023/ |
45 |
0,575 |
0,925 |
0,625 |
1,48 |
1,61 |
Cluster_4_end |
47,5 |
0 |
0,925 |
0,575 |
1,64 |
- |
Gel plan:
Stain |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
Tube number |
|
|
A1 |
A2 |
A3 |
A4 |
|
B1 |
B2 |
B3 |
B4 |
Componants |
Ladder |
|
|
|
|
|
|
|
|
|
|
|
Concentration (ng/µL) |
OD(230nm) |
OD(260nm) |
OD(280nm) |
OD(260nm)/OD(280nm) |
OD(260nm)/OD(230nm) |
Cluster 4 start |
105 |
0,3 |
2,1 |
1,1 |
1,79 |
2,40 |
/936011 |
55 |
0 |
1,1 |
0,55 |
1,99 |
- |
936011/936023 |
520 |
4,325 |
10,425 |
5,825 |
1,79 |
4,38 |
936023/ |
47,5 |
0 |
0,925 |
0,5 |
1,89 |
- |
Cluster 4 end |
130 |
0,6 |
2,625 |
1,475 |
1,79 |
4,38 |
DNA concentration assay
pNP-Assay
- Culture of DH5-alpha transformed with 808030 in pDG011
- Enzymatic essay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5α
Gibson Assembly :
Enzymatic digest of K936011, K936023 and K1392932
- Enzymatic digest using Pst I and Spe I
- Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0,8% Agarose Gel
- Gel Purification
Cluster 4A Gibson assembly :
- Gibson Assembly
- PCR amplification of cluster 4A Assembly.
- Gel migration on 0,8% Agarose Gel.
Gel Plan:
Well Number |
1 |
2 |
3 |
4 |
5 |
Contains |
DNA ladder |
No primers |
Primer for only |
Primer rev only |
Both primers |
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