Team:Groningen/Notebook/tasA Digestion t19
Blue Bio Energy
tasA Digestion (t19)
tasA in the RFP backbone was digested to check for right insert.
Our goal is to overexpress TasA by adding a second copy of the tasA gene under the control of a salt inducible promoter. This overexpression will lead to a stronger biofilm when the biofilm is exposed to salt water.
By digestion it was found out that the transformation of tasA in the RFP backbone failed. Do not use the RFP backbone for further experiments.
Restriction
00:00, 27 May 2015 - 00:00, 27 May 2015
The miniprep product was digested with EcoRI and PstI to check for insert.
Sample
1
2
3
4
5
\( \mathrm{H_2O}\)
14 µL
15 µL
15 µL
15 µL
15 µL
EcoRI
1 µL
1 µL
1 µL
1 µL
1 µL
PstI
1 µL
1 µL
1 µL
1 µL
1 µL
Sample DNA
2 µL
1 µL
1 µL
1 µL
1 µL
10x buffer (2.1)
2 µL
2 µL
2 µL
2 µL
2 µL
Digestion samples.
The samples were incubated at 37 °C for 10 minutes. Afterwards a gel was run with 1% agarose and EtBr.
For each sample:
2 µL miniprep sample
1 µL 6x buffer
3 µL \( \mathrm{H_2O}\)
Ladder:
3 µL GeneRuler™ 1 kb DNA Ladder
On the gel, multiple bands were visible per plasmid, so the plasmid seems to be wrong. Again tasA was gelpurified and ligated into the backbone. Also the backbone (BB1) was checked by the next steps. The following combinations were made:
EcoRI + PstI + RFP plasmid
EcoRI + SpeI + RFP plasmid
XbaI + PstI + RFP plasmid
XbaI + SpeI + RFP plasmid
uncut RFP plasmid
Digestion Mastermix:
12 µL 10x buffer (2.1)
9 µL DNA (RFP plasmid)
99µL \( \mathrm{H_2O}\)
Per sample:
28 µL Mastermix
2 µL restriction enzymes
The samples were incubated for 30 minutes at 37 °C. Afterwards the samples were loaded on gel, 1% agarose with EtBr.
For each sample:
5 µL sample
1 µL 6x buffer
Ladder:
2 µL GeneRuler™ 1 kb DNA Ladder
On the gel it was seen that BB1 is probably not good, as it still has RFP in it. This was checked with the restriction enzymes of Molgen.
Component
Amount
10x buffer (2.1)
6.6 µL
EcoRI
2.2 µL
PstI
2.2 µL
\( \mathrm{H_2O}\)
50.6 µL
Components for Mastermix digestion.
Component
Amount
RFP plasmid DNA
2 µL
10x buffer
3 µL
EcoRI (Molgen)
1 µL
PstI(Molgen)
1 µL
\( \mathrm{H_2O}\)
23 µL
Components for digestion.
The mixture was incubated at 37 °C for 2 hours. After 2 hours the sample was loaded on a 1% agarose gel with EtBr gel with uncut plasmid. On the gel it was visible that the band of the digested plasmid is lower, but there was no insert visible. Do not use this backbone for further ligations and transformations.