Team:Paris/July 28

From 2008.igem.org

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== Attempt to excise and purify the B0034 and B0030 BioBricks ==
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== Purification of the B0034 and B0030 BioBricks ==
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We performed PCR on the B0034 BioBrick (MP 100) and the B0030 BioBrick (MP 120) to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
+
''We performed PCR on the B0034 BioBrick (MP 100) and the B0030 BioBrick (MP 120) to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.''
=== PCR Protocol ===
=== PCR Protocol ===
-
For each samples,  
+
''For each samples,''
* 1 µl dNTP
* 1 µl dNTP
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* 50 µl qsp H2O (33µl)
* 50 µl qsp H2O (33µl)
-
When the PCR cycles were finished, 10 µL of 6X loading dye were added. The samples were then loaded (2 x 30 µL per sample) on a 1,5% agarose gel. After electrophoresis, the bands corresponding to MP 100 and MP 120 were excised and purified using the QIAquick DNA Gel Extraction Kit (QIAGEN). The elution was made in 50 µL of water. Because the intensity of the band corresponding to MP 120 was very low, we only continued with MP 100. MP 100 was digested by EcoRI & SpeI (Forward Insert) or by XbaI & PstI (D100 : Backward Insert).
+
 
 +
=== Electrophoresis - Purification ===
 +
 
 +
''When the PCR cycles were finished,''
 +
 
 +
* Add 10 µL of 6X loading dye.
 +
* Load the samples (2 x 30 µL per sample) on a 1,5% agarose gel.  
 +
* Migration 30' at 100W.
 +
 
 +
 
 +
''After electrophoresis,''
 +
 
 +
* Excision and purification of the bands corresponding to MP 100 and MP 120, using the QIAquick DNA Gel Extraction Kit (QIAGEN).  
 +
* Elution in 50 µL of water.  
 +
 
 +
''Because the intensity of the band corresponding to MP 120 was very low, we only continued with MP 100.''
 +
 
=== DNA Digestion ===
=== DNA Digestion ===
-
Digestion reaction (total volume : 50 µL)
+
''MP 100 was digested by EcoRI & SpeI (Forward Insert) or by XbaI & PstI (D100 : Backward Insert)
 +
 
 +
Digestion reaction (total volume : 50 µL)''
 +
 
* 25 µL DNA
* 25 µL DNA
* 5 µL buffer 2 (10X)
* 5 µL buffer 2 (10X)
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* 15,5 µL water
* 15,5 µL water
-
The reaction was incubated 2 hours at 37°C. The samples (2 x 30 µL per sample) were then analysed by electrophoresis.
+
* The reaction was incubated 2 hours at 37°C.
 +
* The samples (2 x 30 µL per sample) were then analysed by electrophoresis.
 +
 
===Electrophoresis===
===Electrophoresis===
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Revision as of 16:14, 5 August 2008

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Contents

DNA Extraction

Results of the extraction : Electrophoresis

conditions of electrophoresis:

  • 10µl of ladder 1 kb
  • xµl of extraction added with 2µl of loading Dye 6x
  • migration ~30min at 100W


  • Gel 1, 2, 3, 4 = 0,8%
  • 2µl of extraction added with 2µl of loading Dye 6x
  • Gel 5, 6 = 0,8%
  • 3µl of extraction added with 2µl of loading Dye 6x
  • Gel 7 = 0,8%
  • 5µl of extraction added with 2µl of loading Dye 6x
  • Gel 8 = 1,5%
  • 10µl of extraction added with 2µl of loading Dye 6x


Results :

gel1 gel1 gel2 gel2 gel3 gel3 gel4 gel4 gel5 gel5


gel6 gel6 gel7 gel7 gel8 gel8



Name BioBrick Tube N° Enz 1 Enz 2 Obs Exp Size Mea Size Conc (ng/µl) Gel Band
D100 B0034 1 XbaI PstI BI 34 pb - - 8 5
2 - - -
D101 B0034 3 EcoRI XbaI FV 2076 pb - & 2000 pb - & 7 1 & 7 2 & 2
D102 B0034 4 SpeI PstI BV 2077 pb 2000 pb - & 5 1 & 7 3 & 3
5 - & 10 1 & 7 4 & 4
D103 J23101 1 SpeI PstI BV 2100 pb 2000 pb - 1 5
2 10 1 6
D104 J23109 1 SpeI PstI BV 2100 pb 2000 pb 20 1 7
2 20 1 8
D105 R0079 1 SpeI PstI BV 2222 pb 2000 pb 5 2 2
2 5 2 3
D106 R0040 1 SpeI PstI BV 2119 pb - & 2000 pb - & 5 2 & 7 4 & 5
2 - & 8 2 & 7 5 & 6
D107 S03154 1 SpeI PstI BV 2750 pb - & 3000 pb - & 5 2 & 7 6 & 7
D108 S03154 2 XbaI PstI BI 707 pb - & - - & - 2 & 7 7 & 8
3 - & - 2 & 7 8 & 9
D109 S03154 4 EcoRI SpeI FI 708 pb - & - - & - 3 & 7 2 & 10
D110 S03879 1 SpeI PstI BV 2768 pb 3000 pb 20 3 3
D111 S03879 2 XbaI PstI BI 725 pb - - 3 4
3 - 3 5
D112 S03879 4 EcoRI SpeI FI 726 pb - - 3 6
D113 C0079 1 EcoRI SpeI FI 779 pb 800 pb 20 3 7
D114 C0079 2 XbaI PstI BI 778 pb 800 pb 20 3 8
D115 C0179 1 EcoRI SpeI FI 746 pb - & - - & - 4 & 7 2 & 11
D116 C0179 2 XbaI PstI BI 745 pb - & - - & - 4 & 7 3 & 12
D117 E0030 1 EcoRI SpeI FI 746 pb 700 pb 5 4 4
D118 E0030 2 XbaI PstI BI 745 pb 700 pb 5 4 5
D119 E0040 1 EcoRI SpeI FI 743 pb - & 800 pb - & 10 4 & 7 6 & 13
D120 E0040 2 XbaI PstI BI 742 pb - & 800 pb - & 10 4 & 7 7 & 14
D121 E1010 1 EcoRI SpeI FI 704 pb 700 pb 5 4 8
D122 E1010 2 XbaI PstI BI 703 pb 800 pb 10 5 2
D123 J23100 1 SpeI PstI BV 2100 pb 2000 pb 10 5 3
2 10 5 4
D124 J23107 1 SpeI PstI BV 2100 pb 2000 pb 10 5 5
2 15 5 6
D124 J23107 1 EcoRI XbaI FV 3303 pb 3000 pb 15 5 7
2 15 5 8
D126 I0500 1 SpeI PstI FV 5621 pb 6000 pb 10 5 9
2 10 5 10
D127 B0030 1 XbaI PstI BI 37 pb - & - - & - 8 3
2 - - -
D128 B0030 3 EcoRI XbaI FV 2079 pb 2000 pb 10 5 11
D129 B0030 4 SpeI PstI BV 2080 pb 2000 pb 10 5 12
5 10 5 13
D130 E0422 1 XbaI PstI BI 939 pb 1100 pb 10 5 14
2 10 5 15
3 10 5 16
D131 E0840 1 XbaI PstI BI 900 pb 1000 pb 5 6 3
2 10 6 4
3 5 6 5


==> Conclusion : for most of the samples we have enough signal to determine the concentration of the digestion to do the ligation.


For the samples we don't succeed to obtain signals, we have migrated a second gel ( gel n°7).


==> Conclusion : The gel n°7 allowed us to know the concentration of other digestion undetermined.



Ligation

Protocol

For each samples,

  • 1 µl Ligase
  • X µl Vector
  • Y µl Insert (3x more than vector)
  • 2 µl Ligase Buffer 10x
  • 20 µl qsp H2O


  • Incubates O/N at 4°C (in the fridge)


Name Vector Insert Antibio Vol. vector (µl) Vol. insert (µl) Vol. H20 (µl)
L100 D110 D130 A 3 6 8
L101 D110 D131 A 3 6 8
L102 D129 D118 A 5 10 2
L103 D129 D122 A 5 5 7
L104 D129 D114 A 5 4 8
L105 D123 D130 A 5 4 8
L106 D123 D131 A 5 8 4
L107 D103 D130 A 5 8 4
L108 D103 D131 A 5 8 4
L109 D124 D130 A 4 7 6
L110 D124 D131 A 4 7 6
L111 D104 D130 A 3 5 9
L112 D104 D131 A 3 5 9
L113 D126 D130 K 5 2.5 9.5
L114 D126 D131 K 5 2.5 9.5
L115 D105 D130 A 7 11 -
L116 D105 D131 A 7 11 -
L117 D125 D117 A 4 3 10
L118 D125 D121 A 4 2 11
L119 D125 D113 A 4 2 11
L120 D106 D130 A 6 7.5 3.5
L121 D106 D131 A 6 7.5 3.5
L122 D107 D130 A 7 3.5 6.5
L123 D107 D131 A 7 3.5 6.5
L124 D102 D122 A 6 5 4
L125 D102 D1118 A 6 10 1
L126 D102 D114 A 6 4 7
L127 D125 D119 A 6 3 8
C1 D110 - A 3 - 14
C2 D129 - A 5 - 12
C3 D123 - A 5 - 12
C4 D103 - A 8 - 9
C5 D124 - A 7 - 10
C6 D104 - A 3 - 14
C7 D126 - A 2.5 - 14.5
C8 D105 - A 11 - 6
C9 D125 - A 2 - 15
C10 D106 - A 7.5 - 9.5
C11 D107 - A 3.5 - 13.5
C12 D102 - A 10 - 7

Purification of the B0034 and B0030 BioBricks

We performed PCR on the B0034 BioBrick (MP 100) and the B0030 BioBrick (MP 120) to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

PCR Protocol

For each samples,

  • 1 µl dNTP
  • 10 µl Buffer Phusion 5x
  • 2,5 µl VR2 (O18)
  • 2,5 µl VF (O19)
  • 1 µl Phusion
  • 50 µl qsp H2O (33µl)


Electrophoresis - Purification

When the PCR cycles were finished,

  • Add 10 µL of 6X loading dye.
  • Load the samples (2 x 30 µL per sample) on a 1,5% agarose gel.
  • Migration 30' at 100W.


After electrophoresis,

  • Excision and purification of the bands corresponding to MP 100 and MP 120, using the QIAquick DNA Gel Extraction Kit (QIAGEN).
  • Elution in 50 µL of water.

Because the intensity of the band corresponding to MP 120 was very low, we only continued with MP 100.


DNA Digestion

MP 100 was digested by EcoRI & SpeI (Forward Insert) or by XbaI & PstI (D100 : Backward Insert)

Digestion reaction (total volume : 50 µL)

  • 25 µL DNA
  • 5 µL buffer 2 (10X)
  • 2 µL enzyme 1
  • 2 µL enzyme 2
  • 0.5 µL BSA (100X)
  • 15,5 µL water
  • The reaction was incubated 2 hours at 37°C.
  • The samples (2 x 30 µL per sample) were then analysed by electrophoresis.


Electrophoresis

1 2 3 4 5 6
ladder EcoRI & SpeI EcoRI & SpeI - XbaI & PstI XbaI & PstI
Image gel.jpg

The sequence of MP 100 (B0034) digested by EcoRI & SpeI (35 bp) or XbaI & PstI (34 bp) was too short and we didn't manage to visualise it on the gel.

Conclusion : small parts like B0034 can't be cloned as an insert.

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