Template:Team:Groningen/CONTENT/LOGBOOK/tasA Digestion t11
tasA Digestion (t11)
Restriction of tasA to check if the restriction sites are gone.
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.
It was seen that the restriction sites were gone, so the PCR was successful.
<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/restriction">Restriction</a>
00:00, 20 May 2015 - 00:00, 20 May 2015
The gel that was made after the PCR, showed a product at ∼950 bp. This fragment was checked with restriction enzymes to find out if the restriction sites were absent in the tasA gene. This product, PCR product 4.2, was digested with the biobrick assembly kit digestion enzymes (NEB) using EcoRI and PstI. The following samples were made.
#
Compound
Amount
1
Buffer 10x
2 µL
EcoRI
1 µL
PCR prod 4.2
2 µL
\( \mathrm{H_2O}\)
15 µL
2
Buffer 10x
2 µL
PstI
1 µL
PCR prod 4.2
2 µL
\( \mathrm{H_2O}\)
15 µL
Components used for digestion of PCR product 4.2.
The following digestion protocol was followed. See Table 3.
#
Step
Temperature
Time
1
Digestion
37 °C
30:00
2
Enzyme inactivation
80 °C
10:00
Digestion protocol.
A gel was loaded with digestion product of sample 1 and 2 on 1% agarose with EtBr. The following components were added to each well.
Sample:
1.5 µL 6x loading buffer
5 µL digestion product
Ladder:
3 µL GeneRuler™ 100 bp DNA Ladder
On the gel there was one band visible for sample 1 at ± 950 bp. Apparently EcoRI did not cut tasA. A mutation in the EcoRI restriction site of tasA was possibly present. The gel of sample 2 showed a product at ± 950 bp, so pstI did not cut tasA. So a mutation in the PstI restriction site of tasA might have been present.