Difference between revisions of "GDSYZX-United/NOTEBOOK/protocol"

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                                     <p><div class="m-b-xs text_font"><strong>7.RT-PCR</strong></div></p>
 
                                     <p><div class="m-b-xs text_font"><strong>7.RT-PCR</strong></div></p>
 
                                     <p><div class="text_font">Experiment macterial：M-MuLV</div></p>
 
                                     <p><div class="text_font">Experiment macterial：M-MuLV</div></p>
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<p><div class="text_font">(1)preparation for RT-PCR</div></p>
 
 
<p><div class="text_font">(1)preparation for RT-PCR</div></p>
 
<p><div class="text_font">(1)preparation for RT-PCR</div></p>
 
<ul>
 
<ul>

Revision as of 14:39, 16 October 2016

Protocol

1.Extract Arabidopsis genomic DNA

Material:

  • EZ-10 Spin Column Plant Genomic DNA Purification Kit

  • β-mercaptoethanol

  • RNaseA

  • Chloroform

  • Ice box and ice

2.PCR

Material:

  • ddH20

  • dNTPs mix

  • 10×Buffer

  • Taq polymerase

  • PCR Primers

  • Arabidopsis genomic DNA

Methods:

  1. add material into a 0.2ml EP tube according to the table

    Material dosage/μl
    dNTPs mix 1
    Forward primer 0.5
    Reverse primer 0.5
    Arabidopsis genomic DNA 3
    ddH20 12
    Taq polymerase 0.5
    Total 20

  2. set the PCR program

    • For promoter cop1,phyB,pif1,gene hhl1,gene flu:

      94℃ × 5min +(94℃×30s +Tm×30s +72℃×1min)×35+72℃×5min +4℃(for preserving)

    • For pHhl1-gene hhl1:

      94℃ × 5min +(94℃×30s +Tm ×30s +72℃×90s) ×30+72℃×5min +4℃(for preserving)

    • Tm of each primer:

      Name Tm(Forward/Reverse)(℃) Tm in PCR(℃)
      Promoter pif1 62.59/62.67 63
      Promoter cop1 66.77/65.46 66
      Promoter phyB 63.94/63.90 64
      pHhl1-genehhl1 60.75/59.38 60
      gene hhl1 60.90/59.38 60
      Fluorescent gene flu 62.42/66.90 65

3.digestion

Material:

  • ddH20

  • BsaⅠBuffer

  • PstⅠBuffer

  • restriction enzyme BsaⅠ，PstⅠ，EcorⅠ

  • PstⅠBuffer:100mM NaCl、50mMTris-Hcl、10mM MgCl2、100μg/mlBSA、ph7.9

  • Bsa1 Buffer:50mM CH3COONa、20mMTris-Hcl、10mM(CH3COO)2Mg、100μg/mlBSA、ph7.9

Method

  1. add materials into a 1.5ml EP tube according to the table below:

    2. Material dosage/μl
    PCR product(after purification) 17
    restriction enzyme buffer 3 (/4)
    restriction enzyme 1(0.5 each)
    ddH20 4
    Total 25

    PCR product cutting sites restriction enzyme Buffer
    pHhl1-hhl1 BsaⅠ+EcorⅠ 3μlPstⅠ+1μlBsaⅠ
    hhl1 PstⅠ+EcorⅠ 3μlPstⅠ
    flu EcorⅠ+BsaⅠ 3μlPstⅠ+1μlBsa1Ⅰ
    cop1 BsaⅠ+PstⅠ 3μlPstⅠ
    phyB BsaⅠ+PstⅠ 3μlPstⅠ
    pif1 BsaⅠ+PstⅠ 3μlPstⅠ

  2. Place the tube in a 37℃ incubator, stand for 1 hour.

  3. Water bath the tube in 65℃ for 20 mins to end the digestion reaction.

4.chemical conversion

(1)preparation of LB Broth

  • 2g peptone

  • 1g NaCL

  • 0.2g glucose

  • 1g yeast

  • Add water to 200ml, regulate the pH at a range of 6.8~7.2

Add water to 200ml, regulate the pH at a range of 6.8~7.2

(2)preparation of LB Broth Medium

(3)Preparation of chloromycetin solution

  • 10ml ddH20

  • 0.25g Chloromycetin

Add trace amount of NaOH until the chloromycetin dissolves completely

(4)add chloromycetin solution into LB broth, mix them

(5)Add competence DH5α （100μl）into 10 μl plasmid，mix them gently, and ice bath

(6)Heat the solution in 42℃ water bath，and ice-bath 2min (Be sure not to shake the centrifuge tube)

(7)Add 890μl, 37℃ preheating LB broth, mix them gently ,shake them in the condition 37℃, 150rpm for 45min.

(8)4000rpm centrifuge 5min, discard 900 μl supernatant ,suspend the 100μl sediment gentlely

(9)Spread the solution on the LB broth medium evenly, after it is dried, culture it upside down in the 37 ℃ incubator for 16 hours

(10)Put the centrifuge tube which including culture solution and competence DH5α in the bed temperature incubator, shaking culture it in the condition of 220rpm, 37℃ for 16 hours

*THE RESUALT：

(1)The escherichia coli didn't grow on the LB broth medium

(2)After centrifuged, some escherichia colis can be found with a little amount in the bottom of the centrifuge tubes

5.plasmid extraction

Experiment macterial: the kit for plasmid extraction

Experiment apparatus: centrifugal machine, water bath, incubator

Experiment preparation: 60 ℃ water bath， 37 ℃ incubator

(1)centrifuge the bacterial liqiud ,12000rpm 1min. Discard the supernatant.

(2)According to the kit for extraction, extract the plasmid DNA

(3)Preserve the plasmid in 4℃ refrigerator

6.extraction of RNA

Experiment macterial：the kit for RNA fractionation and purification

Experiment steps: refer to protocol

7.RT-PCR

Experiment macterial：M-MuLV

(1)preparation for RT-PCR

  • Buffer 5μl

  • Forward Primer 2μl

  • Reverse primer 2μl

  • RT reverse transcription 0.5μl

  • Taq enzyme 0.5μl

  • RNA 20μl

  • ddH2O 19.5μl

(2)RT-PCR program

45℃×30min+94℃×5min+（94℃×30s+60.5℃×30s+72℃×1min）×40+72℃×10min