User:Saroj Pandey/Notebook/SNP PCR optimization/2014/09/18: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==gDNA extraction from buccal cavity==
==SNP PCR==
'''Primers ending in G'''
*fixed forward 161bp L: high 3' stability


'''Tissue Preparation'''
TAS2R38_1f: GGGATGTAGTGAAGAGGCAGG


1. Gargle with 14 ml 0.9% NaCl. Apply toothbrush to the inner lining of buccal cavity. Collect the tissue suspension in a tube.
Length:  21 bp Tm:  61.0 °C GC:  57.1 % ANY:  2.0 SELF:  0.0


2. Centrifuge at 5000 rpm for 20 minutes.
TAS2R38_1r: GATGGCTTGGTAGCTGTGGT


3. Discard supernatant and transfer pellet in a 1.5 ml eppendorf tube. Add 0.9% NaCl to make up the volume to 1.5 ml.
Length:  20 bp Tm:  60.1 °C GC:  55.0 % ANY:  4.0 SELF:  0.0


4. Centrifuge at 12000 rpm for 4 minutes.
Product Size:  161 bp Pair Any: 3.0 Pair End: 0.0


5. Discard supernatant and use pellet for DNA extraction.


*fixed backward 235bp R: high end self complementarity


'''DNA extraction (Ron's Tissue DNA Mini Kit)'''
TAS2R38_2f: GGTGGCAACCAGGTCTTTAG


1. Add 250 µl buffer PB incl. 20 µl proteinase K. Vortex thoroughly at max. speed for 15 s.
Length:  20 bp Tm:  59.6 °C GC:  55.0 % ANY:  6.0 SELF:  2.0   


2. Incubate the tube at 52°C for 45 min. Vortex occasionally.
TAS2R38_2r: CAATCACTGTTGCTCAGTGC


3. Add 250 µl buffer AB and vortex for 5 s. Transfer solution to a spin column by pipetting.
Length:  20 bp Tm:  58.0 °C GC:  50.0 % ANY:  6.0 SELF:  4.0


4. Centrifuge loaded column at 13000 rpm for 30s. Discard flow-through.
Product Size:  235 bp Pair Any: 5.0 Pair End: 1.0


5. Add 400 µl buffer WB to the spin column. Centrifuge at 13000 rpm for 30s. Discard flow-through.


6. Wash the spin column with 400 µl 70% ethanol by centrifugation at 13000 rpm for 3 min. Carefully remove the tube and discard flow-through.


7. Transfer the column to a 105 ml tube. Place 100 µl buffer EB preheated to 70°C in the centre of the column, close the lid and incubate for 1 min. Centrifuge at 13000 rpm for 1 min to elute DNA.
'''Primers ending in C'''
*fixed forward 161bp


TAS2R38_C1f: GGGATGTAGTGAAGAGGCAGC


Length:21 bp Tm: 61.2 °C GC:57.1 %  ANY:3.0  SELF:3.0 


TAS2R38_C1r: GATGGCTTGGTAGCTGTGGT


'''DNA concentration'''
Length:20 bp  Tm: 60.1 °C GC:55.0 % ANY:4.0 SELF:0.0


1. Taster: 58.3 ng/μL (260:280 - 1.77)


2. Non-taster: 25.7 ng/μL (260:280 - 1.74)
*fixed backward 235bp
 
TAS2R38_C2f: GGTGGCAACCAGGTCTTTAG
 
Length:20 bp Tm: 59.6 °C GC:55.0 % ANY:6.0 SELF:2.0
 
TAS2R38_C2f: CAATCACTGTTGCTCAGTGG
 
Length:20 bp Tm: 57.8 °C GC:50.0 % ANY:6.0 SELF:4.0
 
 
 
'''PCR 1'''
 
[[Image:SNP_PCR1.JPG]]
[[Image:Electrophoresis_GA_11.09.jpg|right|thumb|350px|Electrophoresis]]
[[Image:Electrophoresis_GA_12.09.jpg|right|thumb|350px|Electrophoresis]]
 
'''PCR 2'''
 
[[Image:SNP_PCR2.JPG]]
[[Image:Electrophoresis_GA_16.09.jpg|right|thumb|350px|Electrophoresis]]
 
 
 
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
|}
 
__NOTOC__


==PCR for gDNA amplification==
==PCR for gDNA amplification==

Revision as of 04:20, 19 September 2014

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

SNP PCR

Primers ending in G

  • fixed forward 161bp L: high 3' stability

TAS2R38_1f: GGGATGTAGTGAAGAGGCAGG

Length: 21 bp Tm: 61.0 °C GC: 57.1 % ANY: 2.0 SELF: 0.0

TAS2R38_1r: GATGGCTTGGTAGCTGTGGT

Length: 20 bp Tm: 60.1 °C GC: 55.0 % ANY: 4.0 SELF: 0.0

Product Size: 161 bp Pair Any: 3.0 Pair End: 0.0


  • fixed backward 235bp R: high end self complementarity

TAS2R38_2f: GGTGGCAACCAGGTCTTTAG

Length: 20 bp Tm: 59.6 °C GC: 55.0 % ANY: 6.0 SELF: 2.0

TAS2R38_2r: CAATCACTGTTGCTCAGTGC

Length: 20 bp Tm: 58.0 °C GC: 50.0 % ANY: 6.0 SELF: 4.0

Product Size: 235 bp Pair Any: 5.0 Pair End: 1.0


Primers ending in C

  • fixed forward 161bp

TAS2R38_C1f: GGGATGTAGTGAAGAGGCAGC

Length:21 bp Tm: 61.2 °C GC:57.1 % ANY:3.0 SELF:3.0

TAS2R38_C1r: GATGGCTTGGTAGCTGTGGT

Length:20 bp Tm: 60.1 °C GC:55.0 % ANY:4.0 SELF:0.0


  • fixed backward 235bp

TAS2R38_C2f: GGTGGCAACCAGGTCTTTAG

Length:20 bp Tm: 59.6 °C GC:55.0 % ANY:6.0 SELF:2.0

TAS2R38_C2f: CAATCACTGTTGCTCAGTGG

Length:20 bp Tm: 57.8 °C GC:50.0 % ANY:6.0 SELF:4.0


PCR 1

Electrophoresis
Electrophoresis

PCR 2

Electrophoresis



PCR for gDNA amplification

Product Size: 992 bp Pair Any: 6.0 Pair End: 2.0

TAS2R38_gaF: ATCCGTGATGCTGTGCTATG

Length: 20 bp Tm: 59.7 °C GC: 50.0 % ANY: 4.0 SELF: 0.0

TAS2R38_gaR: GCATCCCAGAAGAAACCAGA

Length: 20 bp Tm: 60.2 °C GC: 50.0 % ANY: 2.0 SELF: 0.0


PCR 1


Agarose gel electrophoresis

  • 0.7% agarose gel prepared
  • 700 mg of agarose was added to water and heated at 600 °C for 90 seconds
  • 10 mg gel red was added and mixed
  • the gel mixture was added to the casting apparatus and comb was placed
  • 450 ml TAE buffer was poured to the electrophoretic chamber
  • After the gel was set, it was transferred to the electrophoretic chamber
  • the comb was slowly taken out
  • Sample mixtures (5μL water + 2μL loading dye + 5μL PCR product / 2μL DNA ladder) were loaded to individual wells and the chamber was covered
  • electrophoresis was run at 120 V for 30 minutes
Electrophoresis


Observations

  • first few samples including 100 bp ladder are invisible
  • 1 kb ladder does not show distinct bands
  • All the PCR products are seen below 250bp and are diffused
  • Desired region of the genomic DNA was not amplified



PCR 2

  • Amount of Phusion polymerase was changed
  • Annealing temperature was decreased
  • Taq DNA polymerase was also used

Electrophoresis
Electrophoresis

Agarose gel electrophoresis

  • 0.7% agarose gel prepared
  • 700 mg of agarose was added to water and heated at 600 °C for 90 seconds
  • 10 mg gel red was added and mixed
  • the gel mixture was added to the casting apparatus and comb was placed
  • 450 ml TAE buffer was poured to the electrophoretic chamber
  • After the gel was set, it was transferred to the electrophoretic chamber
  • the comb was slowly taken out
  • (5μL water + 2μL loading dye + 5μL PCR product / 2μL DNA ladder)

Sample mixtures were loaded to individual wells and the chamber was covered

  • electrophoresis was run at 120 V for 30 minutes



Observation

  • Phusion polymerase could not amplify the desired region of DNA
  • Two bands were observed for Taq polymerase: one at about 1000bp and the other below 250bp
  • Amplifiction was successful with taq DNA polymerase
  • however, the bands are unclear and diffused
  • reason for unclear and diffused bands may be the composition of agarose gel as it was prepared just in water


Agarose gel electrophoresis (repeated)

  • agarose gel was prepared in TAE buffer and the samples were run


Observation

  • bands look much clear and distinct
  • PCR products of Taq DNA polymerase show bands at 1kbp which was estimated (992 bp)
  • blank with Phusion DNA polymerase shows a thin band and that with Taq DNA polymerase shows a discinct and below 100 bp. These bands could not be primer dimers as they could only be as long as 40bp.
  • unspecific bands/products were also seen in other samples

DNA concentration

1. T3: 520.9 ng/μL (260:280=1.79)

2. N3: 406.7 ng/μL (260:280=1.83)

- Samples T3 and N3 were sent for sequencing


|}


Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Saroj_Pandey/Notebook/SNP_PCR_optimization/2014/09/18&oldid=821971"