Team:Newcastle/3 August 2010

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(New page: {{Team:Newcastle/mainbanner}} =Plasmid Miniprep Experiment= ==Aims== The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''...)
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=Plasmid Miniprep Experiment=
=Plasmid Miniprep Experiment=
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==Aims==
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==Aim==
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The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing ''lacI'' Biobrick from ''E. coli'' DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.
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The aim of this experiment is to extract plasmid DNA pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] from ''E. coli'' DH5α cells using the Qiagen miniprep kit and analysing with the Nanodrop machine.
==Materials and Protocol==
==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol, [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol and [[TeamNewcastleRestriction digests| Restriction digests]] for restriction digestion protocol.
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Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol, [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol and [[TeamNewcastleRestriction digests| Restriction digests]] for restriction digestion protocol. NOTE: 10 µl of RNAse A have been added into the current P1 buffer from Qiagen.
==Result==
==Result==
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[[Image:Picture3.png|300px]]
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'''Figure 1''': Gel electrophoresis of the PCR products
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* '''Lane 1''': 1kb DNA ladder
* '''Lane 1''': 1kb DNA ladder
* '''Lane 2''': Extraction of pSB1C3 plasmid (No. 1)
* '''Lane 2''': Extraction of pSB1C3 plasmid (No. 1)
* '''Lane 3''': Extraction of pSB1C3 plasmid (No. 2)
* '''Lane 3''': Extraction of pSB1C3 plasmid (No. 2)
* '''Lane 4''': Extraction of pSB1C3 plasmid (No. 3)
* '''Lane 4''': Extraction of pSB1C3 plasmid (No. 3)
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* '''Lane 5''': Extraction of pSB1C3 plasmid (No. 4)
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* '''Lane 5''': Extraction of plasmid containing lacI (No. 1)
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* '''Lane 6''': Extraction of plasmid containing lacI (No. 1)
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* '''Lane 6''': Extraction of [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing double terminator (No. 1)
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* '''Lane 7''': Extraction of plasmid containing lacI (No. 2)
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* '''Lane 7''': 1kb DNA ladder
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* '''Lane 8''': Extraction of pSB1AK3 plasmid containing double terminator (No. 1)
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* '''Lane 9''': Extraction of pSB1AK3 plasmid containing double terminator (No. 2)
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{|border=1
{|border=1
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!'''pSB1C3  
!'''pSB1C3  
(No. 3)'''
(No. 3)'''
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!'''pSB1C3
 
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(No. 4)'''
 
!'''lacI  
!'''lacI  
(No. 1)'''
(No. 1)'''
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!'''lacI
 
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(No. 2)'''
 
!'''Double terminator  
!'''Double terminator  
(No. 1)'''
(No. 1)'''
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!'''Double terminator
 
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(No. 2)'''
 
|-
|-
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|44.0 µl/ml
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|92.1 µl/ml
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|19.9 µl/ml
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|110.0 µl/ml
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|25.0 µl/ml
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|110.5 µl/ml
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|30.8 µl/ml
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|246.1 µl/ml
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|10.0 µl/ml
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|246.7 µl/ml
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|44.2 µl/ml
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|9.2 µl/ml
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|39.7 µl/ml
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|}
|}
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'''Table 1''': Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.
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'''Table 1''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
==Discussion==
==Discussion==
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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. One possible explanation for this to happen could be that when the transformed ''E. coli'' DH5α cells were grown overnight for the plasmid extraction protocol, the medium in which they were grown did not contain any antibiotics and because of this the cells did not require plasmid which conferred bacteria with antibiotic resistance and this process is called as plasmid shuffle.
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Bands around 3000 bp were observed in lanes 2, 3 and 4, thereby indicating the correct products. Lane 5 contain the LacI plasmid, therefore it should be bigger in size as compared to the double terminator plasmid which is in lane 6. Therefore lane 5 might not contain the LacI plasmid. From the nanodrop results, the ratio of 260:280 nm ranged from 1.7 to 1.85 and the concentration ranged from 92.1 µl/ml to 246.7 µl/ml. These results indicated that the DNA extracted have low contamination and the samples are pure.
==Conclusion==
==Conclusion==
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This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.
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Extraction of pSB1C3 plasmid and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing the double terminator have been successful. As well as the addition of RNAse into the P1 buffer solved the problem of contamination.
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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 23:18, 27 October 2010

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Contents

Plasmid Miniprep Experiment

Aim

The aim of this experiment is to extract plasmid DNA pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] from E. coli DH5α cells using the Qiagen miniprep kit and analysing with the Nanodrop machine.

Materials and Protocol

Please refer to: Minipreps for Qiagen miniprep protocol, Nanodrop Spectrophotometer for nanodrop protocol and Restriction digests for restriction digestion protocol. NOTE: 10 µl of RNAse A have been added into the current P1 buffer from Qiagen.

Result

Picture3.png

Figure 1: Gel electrophoresis of the PCR products

  • Lane 1: 1kb DNA ladder
  • Lane 2: Extraction of pSB1C3 plasmid (No. 1)
  • Lane 3: Extraction of pSB1C3 plasmid (No. 2)
  • Lane 4: Extraction of pSB1C3 plasmid (No. 3)
  • Lane 5: Extraction of plasmid containing lacI (No. 1)
  • Lane 6: Extraction of [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing double terminator (No. 1)
  • Lane 7: 1kb DNA ladder


pSB1C3

(No. 1)

pSB1C3

(No. 2)

pSB1C3

(No. 3)

lacI

(No. 1)

Double terminator

(No. 1)

92.1 µl/ml 110.0 µl/ml 110.5 µl/ml 246.1 µl/ml 246.7 µl/ml

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

Discussion

Bands around 3000 bp were observed in lanes 2, 3 and 4, thereby indicating the correct products. Lane 5 contain the LacI plasmid, therefore it should be bigger in size as compared to the double terminator plasmid which is in lane 6. Therefore lane 5 might not contain the LacI plasmid. From the nanodrop results, the ratio of 260:280 nm ranged from 1.7 to 1.85 and the concentration ranged from 92.1 µl/ml to 246.7 µl/ml. These results indicated that the DNA extracted have low contamination and the samples are pure.

Conclusion

Extraction of pSB1C3 plasmid and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing the double terminator have been successful. As well as the addition of RNAse into the P1 buffer solved the problem of contamination.

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