Team:Grenoble/Biology/Notebook/August

From 2012.igem.org

(Difference between revisions)
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We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5. Annealing temperature = 65°C.<br/>
We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5. Annealing temperature = 65°C.<br/>
<br/>
<br/>
-
We did some digestions (protocol) on miniprep in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.
+
We did some digestions (protocol) on miniprep in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.<br/>
<br/>
<br/>
To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
Line 78: Line 78:
<h2> Wednesday, August 01<span class="exposant">st</span>:</h2>
<h2> Wednesday, August 01<span class="exposant">st</span>:</h2>
We did a touchdown PCR (from 70°C to 60°C) from miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.<br/>
We did a touchdown PCR (from 70°C to 60°C) from miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.<br/>
 +
<br/>
 +
To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
 +
Migration conditions = 100V during 30 min.<br/>
 +
In order to reveal the DNA fragments, we used EtBr.<br/>
<br/>
<br/>
<center><img src="https://static.igem.org/mediawiki/2012/6/6f/120801.jpg" alt="photo_gel_24"/></center>
<center><img src="https://static.igem.org/mediawiki/2012/6/6f/120801.jpg" alt="photo_gel_24"/></center>

Revision as of 11:15, 13 August 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

August

week 31 week 32 week 33 week 34 week 35

Week 31: July 30th to August 05th

Goal of the week:

We wanted to recover and amplify some biobricks involved in our genetic networks :
  • pSB4C5 (2400bp)
  • pompC (100bp)
  • mcherry (900bp)
  • eCFP (800bp)
  • GFP (100bp)

Tuesday, July 31th:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain with pSB4C5, on which we wanted to recover pSB4C5.

We did some colony PCRs (on 12/07/25 colony) and PCrs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 60°C.

To separate the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_22
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB4C5 (miniprep) PCR product (DMSO)
Lane 3: pSB4C5 (miniprep) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product
Lane 6: pSB4C5 (colony) PCR product (DMSO)
Lane 7: pSB4C5 (colony) PCR product (DMSO)
Lane 8: pSB4C5 (colony) PCR product
Lane 9: pSB4C5 (colony) PCR product
Lane 10: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5. Annealing temperature = 65°C.

We did some digestions (protocol) on miniprep in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_23
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 digestion product
Lane 3: pSB4C5 digestion product
Lane 4: pSB4C5 (miniprep) PCR product (DMSO)
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: pSB4C5 (miniprep) PCR product
Lane 7: pSB4C5 (miniprep) PCR product
Lane 8: pSB4C5 (colony) PCR product (DMSO)
Lane 9: pSB4C5 (colony) PCR product (DMSO)
Lane 10: pSB4C5 (colony) PCR product
Lane 11: pSB4C5 (colony) PCR product
Lane 12: DNA ladder 1kb (biolabs)

For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.
The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the two digestion products (code = 120731AM_DIG_024).

Wednesday, August 01st:

We did a touchdown PCR (from 70°C to 60°C) from miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.

To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_24
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 (digestion) PCR product
Lane 3: pSB4C5 (digestion) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

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