IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/20: Difference between revisions

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(Autocreate 2011/09/20 Entry for IGEM:UNAM_LCG/2009/Notebook/Hydrobium_etli)
 
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==Entry title==
==pBBR1MCS backbone PCR extraction==
* Insert your content here.


===Abstract===
I made a PCR to extract the backbone of pBBR1MCS. The plasmid is standarized so I used the prefix fwd and suffix reverse primers. I need this backbones to insert the entire Anderson collection in R. etli.
I used the plasmids extracted by [[User:Eduardo_Vladimir_Munoz|Vladimir]] (see the previous entrance).
I ran a gel (on 21/09/2011, but I show it in this entrance) to see if the PCRs were well, but unfourtunatly nothing appeared.


===Details===
I ran PCRs from the 4 plasmid extractions made by [[User:Eduardo_Vladimir_Munoz|Vladimir]] to extract the backbone. I used the Invitrogen's™ Platinum® Taq DNA polymerase, to define the reactives consentrations and PCR steps I checked out the manual online for the enzyme: [http://tools.invitrogen.com/content/sfs/manuals/platinumtaq_pps.pdf PCR taq platinum manual].


I made 4 reactions, one for eachplasmid extraction made by [[User:Eduardo_Vladimir_Munoz|Vladimir]]: pBBR1MCS 1, pBBR1MCS 2, pBBR1MCS DH5α and pBBR1MCS S17.
====Reactive ammounts====
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| align="center" style="background:#f0f0f0;"|'''Reactive'''
| align="center" style="background:#f0f0f0;"|'''Ammount (μL)'''
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|--
| MiliQ Water
| 39.5
|--
| Buffer
| 5
|--
| dNTPs 0.4mM
| 1
|--
| MgCl<sub>2</sub>
| 1.5
|--
| PrimerF prefix
| 1
|--
| PrimerR suffix
| 1
|--
| Template DNA
| 0.5
|--
| Taq plat polymerase
| 0.5
|--
| '''Total'''
| '''50'''
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====Reactive details====
* Primers: I used the Prefix-forward and Suffix-reverse primers in order to amplify what was between the preffix and suffix.
* Template DNA: I made a dilution 1 part per 20 of each plasmids before runing the PCR.
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Revision as of 16:35, 21 September 2011

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pBBR1MCS backbone PCR extraction

Abstract

I made a PCR to extract the backbone of pBBR1MCS. The plasmid is standarized so I used the prefix fwd and suffix reverse primers. I need this backbones to insert the entire Anderson collection in R. etli. I used the plasmids extracted by Vladimir (see the previous entrance). I ran a gel (on 21/09/2011, but I show it in this entrance) to see if the PCRs were well, but unfourtunatly nothing appeared.

Details

I ran PCRs from the 4 plasmid extractions made by Vladimir to extract the backbone. I used the Invitrogen's™ Platinum® Taq DNA polymerase, to define the reactives consentrations and PCR steps I checked out the manual online for the enzyme: PCR taq platinum manual.

I made 4 reactions, one for eachplasmid extraction made by Vladimir: pBBR1MCS 1, pBBR1MCS 2, pBBR1MCS DH5α and pBBR1MCS S17.

Reactive ammounts

Reactive Ammount (μL)
MiliQ Water 39.5
Buffer 5
dNTPs 0.4mM 1
MgCl2 1.5
PrimerF prefix 1
PrimerR suffix 1
Template DNA 0.5
Taq plat polymerase 0.5
Total 50

Reactive details

  • Primers: I used the Prefix-forward and Suffix-reverse primers in order to amplify what was between the preffix and suffix.
  • Template DNA: I made a dilution 1 part per 20 of each plasmids before runing the PCR.


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