User:Saroj Pandey/Notebook/SNP PCR optimization/2014/11/14: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav)
 
(One intermediate revision by one other user not shown)
Line 2: Line 2:
|-
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
Line 66: Line 66:
• There were also a few unspecific bands with taster DNA at lower temperatures.
• There were also a few unspecific bands with taster DNA at lower temperatures.


• The most prominent bands for both the taster and the non-taster DNA samples were observed at 60.5°C.
• The most prominent bands for both the taster and the non-taster DNA samples were observed at 58.2°C and 61.5°C.




'''Conclusion'''
'''Conclusion'''


The taster and non-taster alleles can be separated using the two given primer pairs at the optimal temperature of 60.5°C.
Optimal temperature for a PCR to separate the taster and non-taster alleles lies between 58.2°C and 61.5°C.





Latest revision as of 00:32, 27 September 2017

Project name Main project page
Previous entry      

Final separation and gradient PCR

PTC taster and non-taster separation

Two primer pairs were taken so that the PTC taster and non-taster could be separated.

Separation PCR
electrophoresis


Taster specific primer pair:

EP9_F: AGCTATGCCCCCTTTCCTCT

Cfb(R): CAATCACTGTTGCTCAGTGG

Product size: 911bp


Non-taster specific primer pair:

ga_F: ATCCGTGATGCTGTGCTATG

Gfb(R): CAATCACTGTTGCTCAGTGC

Product size: 510bp


Observation

• Taster: Taster specific primer pair produced a 911bp band while there were no specific bands with non-taster specific primer pair.

• Non-taster: Non-taster specific primer pair produced a 510bp band while there were no specific bands with taster specific primer pair.

• Blanks: Water sample used instead of the genomic DNA produced only unspecific products, primer dimers probably, with both sets of primers.


Conclusion

• PTC taster and non-taster can be identified genotypically with PCR using the primer pairs used in this experiment.



Gradient PCR

Gradient PCR using the specific primer pairs was performed to identify optimal temperature for both taster and non-taster.

gradient PCR
electrophoresis


Observation

• All the temperatures applied in this PCR were able to produce specific bands for both taster and non-taster.

• There were also a few unspecific bands with taster DNA at lower temperatures.

• The most prominent bands for both the taster and the non-taster DNA samples were observed at 58.2°C and 61.5°C.


Conclusion

• Optimal temperature for a PCR to separate the taster and non-taster alleles lies between 58.2°C and 61.5°C.