Team:Newcastle/1 September 2010

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=Gel extraction of amplified plasmid pSB1C3 for ''rocF''=
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=''Bacillus sphaericus'' Rehydration=
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==Aim==
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The aim of this experiment is to perform gel extraction of the band containing the amplified fragment of the plasmid vector pSB1C3.
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==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Gel extraction| Gel extraction]].
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==Result==
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For plasmid vector pSB1C3, we used 1.5% agarose gel and run the gel on 80 Volts for better resolution.
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We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector.
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{|border=1
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|-
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!
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!'''Plasmid Vector pSB1C3'''
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|'''Size of the Fragment (in bp)'''
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|2072 approx.
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|}
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'''Table 1''': Table represents the size of the plasmid vector pSB1C3 represented as bands on the gel.
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#Weight of gel 0.3g, nanodrop value 27.3ng/microL
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#Weight of gel 0.4g, nanodrop value 21.6ng/microL
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==Discussion==
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We found a band of the appropriate size and cut it out and placed the part of the gel in a 2ml eppendorf tube.
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==Conclusion==
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Gel electrophoresis was successful and we did gel extraction of the fragment containing plasmid pSB1C3.
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=Natto Rehydration=
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==Aims==  
==Aims==  
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We aimed to rehydrate and plate the natto strain (''Bacillus subtilis ATCC 31578'') bought from DSMZ, Germany. We used Penassay G- Thy Medium to rehydrate the cells.
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We aimed to rehydrate and plate the ''sphaericus'' strain bought from BCCM LMG22257. We used nutrient media to rehydrate the cells. The cells are going to be used as a control for high urease activity.
==Materials==
==Materials==
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#Pennassay G- Thy medium:
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#Nutrient Broth
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*Pennassay broth- 17.5g
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#Nutrient Plates
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*Glucose- 20g
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#Pasteur pipette
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*Thymine- 0.05g
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*Distilled water- 1000ml
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Pennassay broth(per l of water):
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==Procedure==
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*peptone 10g
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Freeze- dried cultures are supplied in vacuum- sealed ampoules.
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*Yeast extract 1.5g
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#Make a small file mark on the ampoule enar the middle of the cotton wool plug.
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*Sodium chloride 3.5g
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#Touch the file mark with a red hot glass rod till the glass cracks.
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*Glucose 1.0g
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#Wait a few seconds to allow air ot enter into the amploule.
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*Potassium phosphate dibasic 3.68g
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#Remove the upper part of the ampoule and the cotton plug.
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*Potassium phospahte monobasic 1.32g
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#Add approximately 0.5ml of nutrient broth with a sterile Pasteur pipette to the dried material after flaming the open end of the ampoule.
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*Beef extract 1.5g
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#Mix the contents gently with the tip of the Pasteur pipette, and transfer the contents to one or more suitable solid and/or liquid media.
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#Allow the cells to rehydrate slowly on solid nutrient media.  
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#It is advisable to include a general medium in order to detect possible air- borne contaminants which may have beem introduced during the opening of the ampoule.
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ph7 store at 2-8oC
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N.B. ''Cultures need at least two times subculturing before they can be optimally used in the experiments''. Also note that this procedure has been provided by the BCCM itself for the successful bacterial rehydration and growth.
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==Conclusion==
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We have cultured the rehydrated cells onto the nutrient agar plates and have put them at 37°C overnight for their optimal growth.
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 01:37, 28 October 2010

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Contents

Bacillus sphaericus Rehydration

Aims

We aimed to rehydrate and plate the sphaericus strain bought from BCCM LMG22257. We used nutrient media to rehydrate the cells. The cells are going to be used as a control for high urease activity.

Materials

  1. Nutrient Broth
  2. Nutrient Plates
  3. Pasteur pipette

Procedure

Freeze- dried cultures are supplied in vacuum- sealed ampoules.

  1. Make a small file mark on the ampoule enar the middle of the cotton wool plug.
  2. Touch the file mark with a red hot glass rod till the glass cracks.
  3. Wait a few seconds to allow air ot enter into the amploule.
  4. Remove the upper part of the ampoule and the cotton plug.
  5. Add approximately 0.5ml of nutrient broth with a sterile Pasteur pipette to the dried material after flaming the open end of the ampoule.
  6. Mix the contents gently with the tip of the Pasteur pipette, and transfer the contents to one or more suitable solid and/or liquid media.
  7. Allow the cells to rehydrate slowly on solid nutrient media.
  8. It is advisable to include a general medium in order to detect possible air- borne contaminants which may have beem introduced during the opening of the ampoule.

N.B. Cultures need at least two times subculturing before they can be optimally used in the experiments. Also note that this procedure has been provided by the BCCM itself for the successful bacterial rehydration and growth.

Conclusion

We have cultured the rehydrated cells onto the nutrient agar plates and have put them at 37°C overnight for their optimal growth.

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