Team:Newcastle/28 July 2010

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(New page: {{Team:Newcastle/mainbanner}} ===Aims=== The aim of this experiment is to prove that ''Bacillus subtilis'' 168 can take up arginine from the media into the cell. ===Materials=== * pH str...)
(→Plasmid Miniprep Experiment)
 
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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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===Aims===
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[[Image:Newcastle_Lab_4.jpeg|260px|right]]
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The aim of this experiment is to prove that ''Bacillus subtilis'' 168 can take up arginine from the media into the cell.
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=PCR Experiment=
 +
 
 +
==Aims==
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The aim of this experiment is to prove that ''Bacillus subtilis'' 168 and 3610 chromosomal DNA extraction worked by amplifying P''araE'' using PCR.
 +
 
 +
[[Image:Newcastle_Thermo.JPG|200px|thumb|right|Thermocycler]]
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[[Image:Newcastle Loading Gel.jpg|200px|thumb|right|Loading the gel]]
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[[Image:Newcastle Prep Chr Gel.jpg|200px|thumb|right|Gel]]
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==Materials and Protocol==
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Please refer to: [[Team:Newcastle/PCR#GoTaq_PCR|GoTaq PCR protocol]].
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==Result==
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[[Image:Gelpic2807.png|300px]]
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'''Figure 1''': Gel electrophoresis of the PCR products
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* '''Lane 1''': 1 Kb DNA ladder
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* '''Lane 2''': ''B. subtilis'' 168 chromosomal DNA containing P''araE''
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* '''Lane 3''': ''B. subtilis'' 168 chromosomal DNA containing P''araE''
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* '''Lane 4''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''
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* '''Lane 5''': ''B. subtilis'' 3610 chromosomal DNA containing P''araE''
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==Discussion==
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We found bands in lanes 2, 3, 4 and 5 of around 200 bp in size which is an approximate size of the P''araE'' which is found on the chromosome of both ''B. subtilis'' 168 and 3610.
 +
 
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==Conclusion==
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This experiment proves that the DNA extraction from both ''B. subtilis'' 168 and 3610 done on 27th July, 2010 was successful.
 +
 
 +
=Plasmid Miniprep Experiment=
 +
 
 +
==Aims==
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The aim of this experiment is to extract plasmid DNA pSB1C3, [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] 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.
 +
 
 +
==Materials and Protocol==
 +
[[Image:Newcastle_overnight_culture.jpg|200px|thumb|right]]
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[[Image:Newcastle_plasmids.jpg|200px|thumb|right]]
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[[Image:Newcastle_pSB1C3.jpg|200px|thumb|right]]
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Please refer to: [[Team:Newcastle/Minipreps| Minipreps]] for Qiagen miniprep protocol and [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for nanodrop protocol.
 +
 
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==Result==
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[[Image:Gelpic28073.jpg|300px]]
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'''Figure 2''': Gel electrophoresis of the plasmid after restriction digestion with EcoR1.
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* '''Lane 1''': 1 kb DNA ladder
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* '''Lane 2''': Extraction of pSB1C3 plasmid
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* '''Lane 3''': Extraction of pSB1C3 plasmid
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* '''Lane 4''': Extraction of plasmid containing ''lacI''
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* '''Lane 5''': Extraction of plasmid containing ''lacI''
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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
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* '''Lane 7''': Extraction of [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing double terminator
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===Materials===
 
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* pH strips
 
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* LB media consisting arginine and ampicillin
 
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* Autopipette
 
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* Gilson pipette
 
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* Autoclaved tips for gilson pipette
 
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* Burnsen Burner
 
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* Universal Tube
 
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===Protocol===
 
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* Set up and label the universal tubes as indicated:
 
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# Negative control- Media not containing ''Bacillus subtilis'' 168
 
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# Test- Media inoculated with ''Bacillus subtilis'' 168 cells
 
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* Unscrew the caps of the universal tubes and after unscrewing, flame the mouth of the tube.
 
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* With the help of an autopipette, pipette out 4ml of LB broth from the stock solution and transfer it into the two universal tubes mentioned above.
 
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* Remove the universal tube consisting of an overnight culture of growing ''Bacillus suntilis'' 168 cells and unscrew the cap and flame the mouth of the tube. 
 
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* Set the Gilson pipette to 1000 µl and attach a autoclaved tip to it.
 
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* Remove 1000 µl out of the tube consisting of overnight culture and put it in the universal tube consisting 4ml of LB media. Do this step twice so as to put 1000 µl in both the control and the test tubes.
 
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* Flame the mouth of the universal tubes and screw the cap (do not over tight the cap so as to allow oxygen to enter the tube)and shake gently so as to suspend the cells equally.
 
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* Put the universal tube on a shaker at 37° C and leave it for the cells to grow.
 
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* Record the pH change of the media after every 1 hour by removing 20 µl of the media from both control and test tubes with the help of Gilson pipette and putting it on the pH strip and note the change in the colour.
 
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===Result===
 
{|border=1
{|border=1
|-
|-
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!Hours
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!'''Lane 1'''
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!Negative control
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!'''Lane 2'''
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!Test
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!'''Lane 3'''
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!'''Lane 4'''
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!'''Lane 5'''
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!'''Lane 6'''
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!'''Lane 7'''
|-
|-
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|0
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|N/A
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|pH 7
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|29.9 µl/ml
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|pH 7
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|28.9 µl/ml
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|-
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|34.0 µl/ml
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|1
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|29.8 µl/ml
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|pH 7
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|6.1 µl/ml
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|pH 7
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|6.7 µl/ml
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|-
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|2
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|pH 7
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|pH 7.5
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|-
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|3
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|pH 7
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|pH 7.5
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|-
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|4
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|pH 7 
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|pH 8
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|-
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|5
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|pH 7
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|pH 8
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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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===Discussion===
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==Discussion==
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Arginine consists of a positive charge on it and thus is acidic in nature. As the cells take up the arginine from the media, the pH of the media increases. The tube labeled Negative control contains no cells and the pH of the media has remained constant for 5 hours which shows that the level of arginine in the media has remained constant, while the tube labeled Test which contains ''Bacillus subtilis'' 168 cells shows an increase in pH which shows that the level of arginine has decreased in the media.
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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.  
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However, the concentration was very low, therefore this will have to be repeated.
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===Conclusion===
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It can be concluded that the ''Bacillus subtilis'' 168 cells are able to take up arginine from the media into the cells.
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 +
==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 the ommission of antibiotic in the media.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 22:28, 27 October 2010

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Newcastle Lab 4.jpeg

Contents

PCR Experiment

Aims

The aim of this experiment is to prove that Bacillus subtilis 168 and 3610 chromosomal DNA extraction worked by amplifying ParaE using PCR.

Thermocycler
Loading the gel
Gel

Materials and Protocol

Please refer to: GoTaq PCR protocol.

Result

Gelpic2807.png

Figure 1: Gel electrophoresis of the PCR products

  • Lane 1: 1 Kb DNA ladder
  • Lane 2: B. subtilis 168 chromosomal DNA containing ParaE
  • Lane 3: B. subtilis 168 chromosomal DNA containing ParaE
  • Lane 4: B. subtilis 3610 chromosomal DNA containing ParaE
  • Lane 5: B. subtilis 3610 chromosomal DNA containing ParaE

Discussion

We found bands in lanes 2, 3, 4 and 5 of around 200 bp in size which is an approximate size of the ParaE which is found on the chromosome of both B. subtilis 168 and 3610.

Conclusion

This experiment proves that the DNA extraction from both B. subtilis 168 and 3610 done on 27th July, 2010 was successful.

Plasmid Miniprep Experiment

Aims

The aim of this experiment is to extract plasmid DNA pSB1C3, [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] 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.

Materials and Protocol

Newcastle overnight culture.jpg
Newcastle plasmids.jpg
Newcastle pSB1C3.jpg

Please refer to: Minipreps for Qiagen miniprep protocol and Nanodrop Spectrophotometer for nanodrop protocol.

Result

Gelpic28073.jpg

Figure 2: Gel electrophoresis of the plasmid after restriction digestion with EcoR1.

  • Lane 1: 1 kb DNA ladder
  • Lane 2: Extraction of pSB1C3 plasmid
  • Lane 3: Extraction of pSB1C3 plasmid
  • Lane 4: Extraction of plasmid containing lacI
  • Lane 5: Extraction of plasmid containing lacI
  • Lane 6: Extraction of [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing double terminator
  • Lane 7: Extraction of [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing double terminator


Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7
N/A 29.9 µl/ml 28.9 µl/ml 34.0 µl/ml 29.8 µl/ml 6.1 µl/ml 6.7 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. However, the concentration was very low, therefore this will have to be repeated.

Conclusion

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 the ommission of antibiotic in the media.

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