Team:Paris/August 5

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Revision as of 14:22, 6 August 2008

Amplification of Promotors of interest (FliA, FliL, FlgA, FlgB, FlhB, FlhDC)

We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

PCR Protocol

Preparation of the templates :--> Resuspend of 1 colony of MG1655 in 100µl of water.

List of Oligos :

Number	Name	Sequence	Length	Comments
O100	FlgA-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT	58
O101	FlgA-R	GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT	58
O102	FlgB-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG	58
O103	FlgB-R	GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT	58
O108	FlhB-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG	58
O109	FlhB-R	GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC	58
O111	FlhDC-Total-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTCATTTTTGCTTGCTAGCGTACGGAAAA	58	Amplify the both OmpR binding site
O113	FlhDC(nu)-R	GTTTCTTCCTGCAGCGGCCGCTACTAGTACAGAATAACCAACTTTATTTTTATG	54	Don't amplify the natural rbs of FlhD (only promoter)
O124	FliL-F	GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCAGCGAGAGGCTGTTGGTATTAATGACT	58
O125	FliL-R	GTTTCTTCCTGCAGCGGCCGCTACTAGTACCAGCGATGAAATACTTGCCATGCGATTT	58

Preparation of PCR mix :

For each samples,

1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)

Program PCR: Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Electrophoresis Purification of PCR

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.

gel 1 gel 2

Name	Promotor	Gel	Band	Expected size	Measured size
PCR_124	pFlgA	1	2-3
PCR_125	pFlgB	1	4-5
PCR_126	pFlhB	2	5-6
PCR_127	pFlhDC	1 & 2	7 & 2

==> Remark : for PCR the negative control (templates = water) can be check on the gel n°2, on the band 3-4

==> Conclusion: for the promotors FlgA, FlgB, FlhB we observe the size expected.
We need to repeat the experiments for the promotors FlhDC.

After electrophoresis, the bands corresponding to the right amplification were excised and purified using the QIAquick DNA Gel Extraction Kit by "Maurice (QIAcube)".
Elution in 50 µL of buffer EB.

@@ Line 98: / Line 98: @@
 * Program PCR: Annealing 55°C - Time élongation 1'30" - Number cycle : 29
 === Electrophoresis Purification of PCR===
@@ Line 105: / Line 107: @@
 * 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 the right amplification 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).
@@ Line 152: / Line 149: @@
 |align="center"|
 |}
@@ Line 161: / Line 157: @@
 <br>We need to repeat the experiments for the promotors '''FlhDC'''.
-=== DNA Digestion ===
-''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
-* Incubate 2 hours at 37°C
-* The samples (2 x 30 µL per sample) were then analysed by electrophoresis.
-===Electrophoresis (bis)===
-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.
+* After electrophoresis, the bands corresponding to the right amplification were excised and purified using the QIAquick DNA Gel Extraction Kit by "Maurice (QIAcube)".
+* Elution in 50 µL of buffer EB.