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/> | ||
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<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
August
week 31 week 32 week 33 week 34 week 35Week 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.
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)
(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.
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)
(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.
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)
(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.