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PCR

lrob lrob
wrote...
Posts: 30
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13 years ago Edited: 13 years ago, bio_man
I am asked to work out the size of the product of a PCR reaction, I am not sure how to do this and I would appreciate any help- if its quite a long process, then I would appreciate any guidelines on how to do this.
 
I have two primers- a forward (ACTCTCACTGCTAAGGACA) and reverse (TGGCCATGACCACAAGGATG), which are designed to amplify a section of a cloned gene. If a PCR reaction is ran, what is the expected size of the product in base-pairs, if the DNA sequence is: TACACACAACATGTCCA
CTCTCACTGCTAAGGACACGGAAACAGTC AAGGCCTTCTGGGCCAAAGTGGCTGGAAA GC
CGCAGGACATCGGTGCCGCTGCTCTGAAC AGGATGCTGGTCGTGTACCCGCAGACCAA GACTTACTTCTCCAACTGGCCGGACCAGA GCC
CCAACCCTGCTGAGGTCAGGAAGCACGAA ATGACCGTCATGGTGGGAGTTGAGTATGC TGTGACCAAAATCGACGACCTGAAAGAAG GTCGAAAGAGACTCAGT
CAGCTGCATGCCTTCACCCTGAGAGTGGA CCCTGCCAACTTCAAGATCCTCTCCCACA GCATCCTTGTGGTCATGGCCATCAGGTTC CCAGCCGAC
TTCACCGCTGAGATCCATGTGTCTACGGA CAAGTTCCTGGCAGCTGTGGCTCTGGCCC TGGCTGAGCAGTACAGATCAACAAAAGGA GAAGACGACG
GAGTCTGCTTCTCCAGCAGCCTACTGAAT AATAAATGAATGA

The above DNA sequence was derived from an RNA sample, and reverse transcribed to make a cDNA clone and then subjected to the PCR.

Also if this cDNA had not been reverse transcribed, and it was subjected to gel electrophoresis, and had the same band pattern as an RNA sample that had been reverse transcribed, then what conclusions might be reached?

Thank you very much
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3 Replies
Replies
wrote...
Educator
13 years ago
Hey Irob, this looks complicated.

Quote
What is the expected size of the product in base-pairs, if the DNA sequence is...

I believe this requires some BLAST work, though I'm not totally sure Undecided.

Check this tutorial: http://www.ctbiobus.org/download/electronicpcr.pdf
http://www.ct.gov/cen/lib/cen/Genetically_Modified_Organisms_student_guide_v.1.pdf .

There is an example that states "To calculate the exact length of the expected PCR product, subtract the lowest from the highest number and add one" three quarters of the way down... Let me know if you can deduce anything from it.

This site may also be helpful:

http://www.cybertory.org/exercises/primerDesign/index.html

Quote
Also if this cDNA had not been reverse transcribed, and it was subjected to gel electrophoresis, and had the same band pattern as an RNA sample that had been reverse transcribed, then what conclusions might be reached?

That the distance that the DNA/RNA sample travels down the gel is based on the total size of the sample. The cDNA and the RNA sample essentially have the same size and nucleotide sequence. Therefore, electrophoresis discriminates based on the sorting of molecular sizes. So, if two different DNA samples showed an identical pattern and band thickness, you can conclude that they had the same amount of DNA, had fragments of the same size, and had the same DNA molecules.
lrob Author
wrote...
13 years ago
Hello! after much pondering- I think it is simpler than we both thought. I used word processing software to do this. First  I found the forward primer in the DNA code, then I found the reverse complement of th reverse primer which is CATCCTTGTGGTCATGGCCA. So:

TACACACAACATGTCCACTCTCACTGCTAAGGACACGGAAACAGTCAAGGCCTTCTGGGCCAAA GTGGCTGGAAAGCCGCAGGACATCGGT
GCCGCTGCTCTGAACAGGATGCTGGTCGT GTACCCGCAGACCAAGACTTACTTCTCCA ACTGGCCGGACCAGAGCCCCAACCCTGCT GA
GGTCAGGAAGCACGAAATGACCGTCATGG TGGGAGTTGAGTATGCTGTGACCAAAATC GACGACCTGAAAGAAGGTCGAAAGAGACT CAGTC
AGCTGCATGCCTTCACCCTGAGAGTGGAC CCTGCCAACTTCAAGATCCTCTCCCACAG CATCCTTGTGGTCATGGCCATCAGGTTCCCAGC
CGACTTCACCGCTGAGATCCATGTGTCTA CGGACAAGTTCCTGGCAGCTGTGGCTCTG GCCCTGGCTGAGCAGTACAGATCAACAAA AGGAG
AAGACGACGGAGTCTGCTTCTCCAGCAGC CTACTGAATAATAAATGAATGA

Then did a word count of the letters including the primers which came to 314 so it would be 314 bp long.

Thanks for your help!

wrote...
Educator
13 years ago
Incredible, because I tried the same thing and it didn't work. Excellent work. + for you.
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