Team:Newcastle/5 August 2010

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(Solution for the problem)
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==Result==
==Result==
* '''Lane 1''': 1kb DNA ladder
* '''Lane 1''': 1kb DNA ladder
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* '''Lane 2''': Extraction of pSB1C3 plasmid (No. 1)
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* '''Lane 2''': BioBrick compatible vector pSB1C3
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* '''Lane 3''': Extraction of pSB1C3 plasmid (No. 2)
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* '''Lane 3''': Pspac_oid promoter
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* '''Lane 4''': Extraction of pSB1C3 plasmid (No. 3)
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* '''Lane 4''': 1st fragment of ''rocF'' CDS
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* '''Lane 5''': Extraction of pSB1C3 plasmid (No. 4)
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* '''Lane 5''': 2nd fragment of ''rocF'' CDS
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* '''Lane 6''': Extraction of plasmid containing lacI (No. 1)
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* '''Lane 6''': 3rd fragment of ''rocF'' CDS
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* '''Lane 7''': Extraction of plasmid containing lacI (No. 2)
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* '''Lane 7''': Double Terminator
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* '''Lane 8''': Extraction of pSB1AK3 plasmid containing double terminator (No. 1)
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* '''Lane 8''': 1kb DNA ladder
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* '''Lane 9''': Extraction of pSB1AK3 plasmid containing double terminator (No. 2)
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Revision as of 15:43, 5 August 2010

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Contents

Gel Electrophoresis for the Amplified Fragments of rocF

Aim

The aim of the experiment is to perform gel electrophoresis for all the 6 PCR reactions which took place yesterday 4th August, 2010 and thus confirm that all 6 PCR reactions were successful.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

  • Lane 1: 1kb DNA ladder
  • Lane 2: BioBrick compatible vector pSB1C3
  • Lane 3: Pspac_oid promoter
  • Lane 4: 1st fragment of rocF CDS
  • Lane 5: 2nd fragment of rocF CDS
  • Lane 6: 3rd fragment of rocF CDS
  • Lane 7: Double Terminator
  • Lane 8: 1kb DNA ladder
pSB1C3

(No. 1)

pSB1C3

(No. 2)

pSB1C3

(No. 3)

pSB1C3

(No. 4)

lacI

(No. 1)

lacI

(No. 2)

Double terminator

(No. 1)

Double terminator

(No. 2)

44.0 µl/ml 19.9 µl/ml 25.0 µl/ml 30.8 µl/ml 10.0 µl/ml 44.2 µl/ml 9.2 µl/ml 39.7 µl/ml

Table 1: Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.

Discussion

We found bands in the lane 2, 3, 4, 5 and 6 showing the presence of plasmid in E. coli DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. Also while doing nanodrop experiment, we measured 260/280 nm ratio for all the samples came out to be between 2.0 to 2.4 approximately which shows that there is a high RNA contamination in the samples.

Conclusion

This experiment shows that there is plasmid present in the E. coli DH5α cells but they are present in a very low amount and having high RNA contamination possibly due to the following reasons:

  1. P1 buffer which contains RNAse might be contaminated.
  2. RNAse enzyme might have gotten inactive.

Solution for the problem

  1. If P1 buffer of the Qiagen miniprep kit is contaminated, then use a different kit. We have Promega miniprep kit which will be used tomorrow.
  2. If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.



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