User:Jose Fabricio Lopez/Notebook/Logbook/2011/06/20: Difference between revisions

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=Objectives=
=Objectives=
*PCR from [[User:Jose Fabricio Lopez/Notebook/Logbook/2011/06/19|extractions]]
*PCR from [[User:Jose Fabricio Lopez/Notebook/Logbook/2011/06/19|extractions]]
 
*Primers (5 pM/μL) (new set because we are improving new protocol of standardization).
**Primer Forward= CGG GCC CCA TTA TTA TCA TGA CAT TAA CCT ATA AAA ATA GG
***It includes GGGCCC. Site for ApaI.
**Primer Reverse= CGA GCT CGC CTT TTG CTC ACA TCT TCT TTC C
***It includes GAGCTC. Site SacI.
==PCR==
==PCR==
==PCR amplification==
*DNA was obtained from [[User:Jose Fabricio Lopez/Notebook/Logbook/2011/06/19|extractions]] and diluted(2μL DNA in 98μL H<sub>2</sub>O)
===PCR amplification===
{| border="1"
{| border="1"
! Reactives
! Reactives
Line 56: Line 61:
| 50μL
| 50μL
| 50μL
| 50μL
|}
==Control PCR==
{| border="1"
! Reactives
! -R
! -F
! -O
|--
|Buffer Standard Taq 10x NEB 
| 2μL
| 2μL
| 2μL
|--
| Primer F
| 2μL
| 0μL
| 0μL
|--
| Primer R
| 0μL
| 2μL
| 0μL
|--
| .4mM dNTPs
| 10μL
| 10μL
| 10μL
|--
| DNA(Pablo's extraction)
| 2μL
| 2μL
| 2μL
|--
| Taq
| .2μL
| .2μL
| .2μL
|--
| H<sub>2</sub>O
| 3.8
| 3.8
| 3.8
|--
| Total
| 20μL
| 20μL
| 20μL
|}
|}


==Expectatives==
*See in gel amplification of 3 extraction and not amplification in controls.
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__NOTOC__
__NOTOC__

Revision as of 11:09, 28 June 2011

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Objectives

  • PCR from extractions
  • Primers (5 pM/μL) (new set because we are improving new protocol of standardization).
    • Primer Forward= CGG GCC CCA TTA TTA TCA TGA CAT TAA CCT ATA AAA ATA GG
      • It includes GGGCCC. Site for ApaI.
    • Primer Reverse= CGA GCT CGC CTT TTG CTC ACA TCT TCT TTC C
      • It includes GAGCTC. Site SacI.

PCR

  • DNA was obtained from extractions and diluted(2μL DNA in 98μL H2O)

PCR amplification

Reactives Pablo´s extraction Tube 1 Tube 2
Buffer Standard Taq 10x NEB 5μL 5μL 5μL
Primer F 5μL 5μL 5μL
Primer R 5μL 5μL 5μL
10mM dNTPs 1μL 1μL 1μL
DNA 2μL 2μL 2μL
Taq .5μL .5μL .5μL
H2O 31.5 31.5 31.5
Total 50μL 50μL 50μL

Control PCR

Reactives -R -F -O
Buffer Standard Taq 10x NEB 2μL 2μL 2μL
Primer F 2μL 0μL 0μL
Primer R 0μL 2μL 0μL
.4mM dNTPs 10μL 10μL 10μL
DNA(Pablo's extraction) 2μL 2μL 2μL
Taq .2μL .2μL .2μL
H2O 3.8 3.8 3.8
Total 20μL 20μL 20μL

Expectatives

  • See in gel amplification of 3 extraction and not amplification in controls.
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