Template:Team:Groningen/CONTENT/EXPERIMENTS/tasA Digestion (t19)

00:00, 27 May 2015 - 00:00, 27 May 2015

The miniprep product was digested with EcoRI and PstI to check for insert.

\( \mathrm{H_2O}\)

14 µL

15 µL

EcoRI

1 µL

PstI

1 µL

Sample DNA

2 µL

1 µL

10x buffer (2.1)

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.

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.

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.

Harm Ruesink