Team:Newcastle/24 August 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=Miniprep for pGFPrrnB with ''yneA''=
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=Miniprep for pGFPrrnB with filamentous cell part =
==Aims==
==Aims==
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To produce more stocks of vector pGFPrrnB with insert ''yneA''.
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The aim of this experiment is to obtain stock of the filamentous cell part in pGFP-rrnB for transformation into ''Bacillus subtilis'' 168.
==Materials and Protocol==
==Materials and Protocol==
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Please refer to [[Team:Newcastle/Qiagen_Minipreps|qiagen minipreps]] and [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]] for all 12 tubes of miniprep that we did.
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Please refer to [[Team:Newcastle/Qiagen_Minipreps|qiagen minipreps]] and [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]] protocols.
==Results==
==Results==
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The results from the nanodrop:
 
{|border=1
{|border=1
|-
|-
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!DNA
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!'''Tube 1'''
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!Concentration (ng/µl)
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!'''Tube 2'''
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|-
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!'''Tube 3'''
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!1
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!'''Tube 4'''
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!286.1
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!'''Tube 5'''
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|-
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!'''Tube 6'''
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!2
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!'''Tube 7'''
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!304.0
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!'''Tube 8'''
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|-
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!'''Tube 9'''
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!3
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!'''Tube 10'''
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!316.3
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!'''Tube 11'''
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|-
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!'''Tube 12'''
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!4
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!421.6
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|-
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!5
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!518.7
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|-
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!6
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!460.1
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|-
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!7
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!370.1
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|-
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!8
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!377.3
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|-
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!9
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!346.0
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|-
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!10
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!347.4
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|-
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!11
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!202.8
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|-
|-
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!12
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|286.1 µl/ml
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!307.4
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|304.0 µl/ml
 +
|316.3 µl/ml
 +
|421.6 µl/ml
 +
|518.7 µl/ml
 +
|460.1 µl/ml
 +
|370.1 µl/ml
 +
|377.3 µl/ml
 +
|346.0 µl/ml
 +
|347.4 µl/ml
 +
|202.8 µl/ml
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|307.4 µl/ml
|}
|}
 +
 +
'''Table 1''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
==Discussion==
==Discussion==
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The results from the nanodrop showed that we have produced high concentration of vector pGFPrrnB with insert ''yneA''. We will then proceed to digestion.
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The concentration ranged from 286.1 µl/ml to 518.7 µl/ml. Therefore we have obtained high concentration of ''yneA'' in pGFPrrnB.
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=Single and Double Digestion of pGFPrrnB with ''yneA''=
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=Double Digestion of pGFPrrnB with filamentous cell part=
==Aims==
==Aims==
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To check if the insert ''yneA'' has been inserted into vector pGFPrrnB.
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The aim of this experiment is screen our minipreps to check if the insert is present.
==Materials and Protocol==
==Materials and Protocol==
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We are doing two digests for pGFPrrnB and ''yneA'':
We are doing two digests for pGFPrrnB and ''yneA'':
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* Single digest with HinDIII;
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* Single digest with HindIII;
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* Double digest with EcoR1 and Nhe1.
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* Double digest with EcoRI and NheI.
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==Results==
==Results==
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Gel electrophoresis results for digestion:
 
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==Discussion and Conclusion==
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[[Image:Picture5.1.png|400px]]
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=Single digestion of pSB1C3=
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'''Figure 1''': Gel electrophoresis result for restriction digest of pGFPrrnB and ''yneA'' with Nhe1 and Spe1.
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==Aims==
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* '''Lane 1''': 1kb DNA ladder
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* '''Lane 2''': Tube 1
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To linearise the vector pSB1C3. The linear vector will be used as a PCR template for Gibson cloning of the Subtilin immunity and ''rocF'' BioBricks.
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* '''Lane 3''': Tube 2
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* '''Lane 4''': Tube 3
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==Materials and Protocol==
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* '''Lane 5''': Tube 4
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* '''Lane 6''': Tube 5
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We are doing a single digest with HinDIII.
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* '''Lane 7''': Tube 6
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* '''Lane 8''': Tube 7
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Please refer to [[Team:Newcastle/Restriction_digests|restriction digests]] and [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]].
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* '''Lane 9''': Tube 8
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* '''Lane 10''': Tube 9
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==Results==
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* '''Lane 11''': Tube 10
 +
* '''Lane 12''': Tube 11
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* '''Lane 13''': Tube 12
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* '''Lane 14''': 1kb DNA ladder
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Gel electrophoresis results for digestion:
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==Conclusion==
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[[Image:24.08.10.png|500px]]
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The results show that the insert is present, as there is a band at approximately 541bp corresponding to ''yneA'' and a band at approximately 8.4kbp corresponding to pGFPrrnb in lanes 2, 3, 4, 6, 7, 8, 9, 11 and 12.  
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==Discussion and Conclusion==
 
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The digestion was successful — the correct band was seen. We can proceed to gel extraction.
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 02:23, 28 October 2010

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Contents

Miniprep for pGFPrrnB with filamentous cell part

Aims

The aim of this experiment is to obtain stock of the filamentous cell part in pGFP-rrnB for transformation into Bacillus subtilis 168.

Materials and Protocol

Please refer to qiagen minipreps and nanodrop spectrophotometer protocols.

Results

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Tube 10 Tube 11 Tube 12
286.1 µl/ml 304.0 µl/ml 316.3 µl/ml 421.6 µl/ml 518.7 µl/ml 460.1 µl/ml 370.1 µl/ml 377.3 µl/ml 346.0 µl/ml 347.4 µl/ml 202.8 µl/ml 307.4 µl/ml

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

Discussion

The concentration ranged from 286.1 µl/ml to 518.7 µl/ml. Therefore we have obtained high concentration of yneA in pGFPrrnB.

Double Digestion of pGFPrrnB with filamentous cell part

Aims

The aim of this experiment is screen our minipreps to check if the insert is present.

Materials and Protocol

We are doing two digests for pGFPrrnB and yneA:

  • Single digest with HindIII;
  • Double digest with EcoRI and NheI.


Please refer to restriction digests and gel electrophoresis.

Results

Picture5.1.png

Figure 1: Gel electrophoresis result for restriction digest of pGFPrrnB and yneA with Nhe1 and Spe1.

  • Lane 1: 1kb DNA ladder
  • Lane 2: Tube 1
  • Lane 3: Tube 2
  • Lane 4: Tube 3
  • Lane 5: Tube 4
  • Lane 6: Tube 5
  • Lane 7: Tube 6
  • Lane 8: Tube 7
  • Lane 9: Tube 8
  • Lane 10: Tube 9
  • Lane 11: Tube 10
  • Lane 12: Tube 11
  • Lane 13: Tube 12
  • Lane 14: 1kb DNA ladder

Conclusion

The results show that the insert is present, as there is a band at approximately 541bp corresponding to yneA and a band at approximately 8.4kbp corresponding to pGFPrrnb in lanes 2, 3, 4, 6, 7, 8, 9, 11 and 12.


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