From 2013.igem.org

8 July 2013

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Lab 208

Main purpose

Run gel with 1 uL AMO, 10 uL AMO, 20 uL AMO genes from Nitrosomonas europaea. Purification and extraction of the gel for 10 uL AMO. Run 2nd gel with all the sample of 10 uL AMO. Run gel with HAO and CYC genes Nitrosomonas europaea.

Who was in the lab

Kristian, Ariadni, Gosia

Procedure

Run 1st gel (AMO):

1ul of dye
5ul of sample
5ul Kb ladder

Purification of first gel for 10 AMO according to the protocol QIA gel extraction protocol. (given in the Kit)

Run 2nd gel (AMO):

9ul of dye
around 20 ul of each sample
12 ul of ladder

Purification of second gel according to the same protocol.

Measure the concentration of the purified AMO using Nanodrop (pouring together both samples).

Run 3rd gel (HAO,CYC):

1ul of dye
5ul of sample
5ul Kb ladder

Purification of DNA of both HAO and CYC.

Results

Nanodrop measurement:

30.4 ng/ul (AMO)
14.4 ng/ul (HAO)
11.7 ng/ul (CYC)

Pictures from gels

Gel AMO

Wells:

1: 1 AMO
2: 1 AMO
3: 10 AMO
4: 10 AMO
5: 20 AMO
6: 20 AMO
7: 1 Kb Plus DNA Ladder

Conclusions

Conclusion for AMO

The bands for 10 AMO are thick and it is not visible how many base-pairs they have, so gel extraction and purification is needed.

Gel HAO-CYC

Wells:

1: 1 Kb Plus DNA Ladder
2: 3 HAO
3: 6 HAO
4: 10 HAO
5: 3 HAO
6: 6 HAO
7: 10 HAO

Around 3000 base-pairs

8: 3 CYC
9: 6 CYC
10: 10 CYC
11: 3 CYC
12: 6 CYC
13: 10 CYC

Around 1650 base-pairs

14: 1 Kb Plus DNA Ladder

Conclusion for HAO and CYC

The bands have shown that the results are close to 2866 bp for HAO and close to 1497 bp for CYC.

Gel with purified AMO,HAO and CYC

Wells

8: 1 Kb Plus DNA Ladder
11: purified AMO

(Around 3141 bps)

12: purified HAO

(Around 2866 bps)

13: purified CYC

(Around 1467 bps)

Conclusion for Nir

We clearly see band - not the best but not the worst either. The band are over 12kb according to the ladder which does not match up with the around 9102 we expected from the primer design. We rand a blast on the primers to see if there where other possible binding sites in the chromosome. No good match was found so we purified and will run the purification on a new gel.