MDA (Multiple Displacement Amplication): Difference between revisions

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>Sam Chiang
(New page: '''This protocol is modified from Kun: LabNote/Haplotyping/2009-1-21''' ---- ==Reagents== Bottled Nuclease-free water (Ambion) Nuclease free water – for serial dilution (Ambion) 1M ...)
 
>Sam Chiang
 
(4 intermediate revisions by the same user not shown)
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'''This protocol is modified from Kun: LabNote/Haplotyping/2009-1-21'''
'''This protocol is modified from Kun:LabNotes/Haplotyping/2009-1-21[http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/Haplotyping/2009-1-21]'''
----
----


Line 6: Line 6:
  Bottled Nuclease-free water (Ambion)
  Bottled Nuclease-free water (Ambion)
  Nuclease free water – for serial dilution (Ambion)
  Nuclease free water – for serial dilution (Ambion)
  1M Tris HCL (bottled, which company ?)
  1M Tris-HCL (bottled, which company ?)
  pH indicator (testing paper)
  pH indicator (testing paper)


Line 18: Line 18:
  Making fresh 5M KOH every week (WHY need to be fresh?) MW of KOH = 56.11  
  Making fresh 5M KOH every week (WHY need to be fresh?) MW of KOH = 56.11  
  Preparing 1N HCL (the new bottle is 36%~38% = 12 M = 12 N)
  Preparing 1N HCL (the new bottle is 36%~38% = 12 M = 12 N)
  Preparing 1M Tris HCL (the bottled one is 50X, which company ?)
  Preparing 1M Tris buffer (the bottled one is 2X, which company ?)
  Preparing clean 50X and 2X SYBR Green I solution
  Preparing clean 50X and 2X SYBR Green I solution
  Preparing 1 uM N6 primer (diluted from 100 uM)
  Preparing 1 uM N6 primer (diluted from 100 uM)
Line 45: Line 45:
===step3: UV treatment for decontamination of Nucleotide for NS and ALS buffer===
===step3: UV treatment for decontamination of Nucleotide for NS and ALS buffer===
1. In the hood, covering all of gDNA template with black cap first
1. In the hood, covering all of gDNA template with black cap first
2. UV treatment for NS and ALS for 10 min  
2. UV treatment for NS and ALS for 10 min  


Line 60: Line 61:


1. Mix 1 uL template with 1.5 ALS, RT incubation for 3 min
1. Mix 1 uL template with 1.5 ALS, RT incubation for 3 min
2. Transfer to cool block (eppendorf)
2. Transfer to cool block (eppendorf)
3. Adding 16 uL Master mix.
 
4. Adding 1.5 uL NS
3. Adding 1.5 uL NS, and mix completely.
 
4. Adding 16 uL Master mix.
 
5. Cap the tube and quick vortexing and spinning down.
5. Cap the tube and quick vortexing and spinning down.
6. Place into the realtime machine and start the program.
6. Place into the realtime machine and start the program.

Latest revision as of 08:15, 9 February 2009

This protocol is modified from Kun:LabNotes/Haplotyping/2009-1-21[1]



Reagents[edit]

Bottled Nuclease-free water (Ambion)
Nuclease free water – for serial dilution (Ambion)
1M Tris-HCL (bottled, which company ?)
pH indicator (testing paper)

Materials[edit]

strip PCR-tube with caps (2 strip)
1.5 mL tube (x3)
Cleaned Forceps 
Cooled tube rack 

Preparing[edit]

Making fresh 5M KOH every week (WHY need to be fresh?) MW of KOH = 56.11 
Preparing 1N HCL (the new bottle is 36%~38% = 12 M = 12 N)
Preparing 1M Tris buffer (the bottled one is 2X, which company ?)
Preparing clean 50X and 2X SYBR Green I solution
Preparing 1 uM N6 primer (diluted from 100 uM)

Sample[edit]

Human genomic DNA (stored in -20 degree). Stock solution is 10 ng/uL

30 pg/uL = 10 haplotype. 3 pg/uL = 1 haplotype


Procedures:[edit]

step1: Preparing ALS buffer and NS buffer[edit]

1. ALS buffer: Mix 8 uL 5M KOH, 2 uL 0.5 M EDTA, and 10 uL 1 M DTT with 80 uL nuclease-free H2O, quick spin down.

2. NS buffer: Mix 4 mL 1N HCL with 6 mL 1M Tris.HCL (pH = 7.5)

  • Make sure that ALS buffer and NS buffer were made correctly, we test the mixture of them (1:1) with pH meter, the pH of mixture should closed to 7~8.

step2: Preparing the 2 strips of PCR tubes (for two repeats)[edit]

1. Performing serial dilution for template human gDNA into 30 pg/uL, 3 pg/uL, 300 fg/uL, 30 fg/uL and 3 fg/uL.

  • We placed the 45 uL of nuclease-free H2O in PCR tubes first, and transfer 5 uL from the first tube to the second tube.(Transferring: using 20p tips and pippetors; Mixing: using p200 tips and pippetors)

step3: UV treatment for decontamination of Nucleotide for NS and ALS buffer[edit]

1. In the hood, covering all of gDNA template with black cap first

2. UV treatment for NS and ALS for 10 min

step4: Preparing mater mix for 16+2 reactions[edit]

                                1 rxn              18 rxn
H2O                            10.2 uL             183.6 uL
10X Repliphi phi-29 buffer      2.0 uL              36.0 uL
1uM N6 primer                   1.0 uL              18.0 uL
25 nM dNTP                      0.8 uL              14.4 uL
2X SYBR Green I                 1.0 uL              18.0 uL
Repliphi Phi-29 (100 U/uL)      1.0 uL              18.0 uL
                               16.0 uL             288.0 uL  (288/18=16)

step5: Preparing the reactions[edit]

1. Mix 1 uL template with 1.5 ALS, RT incubation for 3 min

2. Transfer to cool block (eppendorf)

3. Adding 1.5 uL NS, and mix completely.

4. Adding 16 uL Master mix.

5. Cap the tube and quick vortexing and spinning down.

6. Place into the realtime machine and start the program.