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Tipranavir Tipranavir
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3 years ago Edited: 3 years ago, Tipranavir
Tried linking images; didn't work...  please see attached images, sorry for the trouble.

A challenge anyone? Does anyone here have extensive experience (and confidence... sigh..) with restriction enzymes, DNA cloning, and using BLAST?

We've been advised to use the resources on www(dot)neb(dot)sg  (it supposedly has many articles/a huge database on restriction enzymes) , but otherwise, we've been left to fend for ourselves... I never knew molecular biology/labwork was this lonely... there's just so much content you have to figure out, alone..  and these aren't even the application questions..

Any input, of any degree, would be much appreciated. Thank you and have a nice day.
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#1
Educator
3 years ago
Hi Tipranavir

Looking at your first question about forward primers, would you happen to have the start sequence for the mouse? Reason I ask is because you're expected to choose the option that complements the anti-sense strand of the mouse. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).

Quote
You want to clone the CDNA for mouse LDHB (NM 008492.3) into pET11-H5alpha. Which of the DNA sequences below could be used as forward primer for this purpose?

a. CCATGGCAACCCTTAAGGAGAAG
b. TGGACACCATGGCAACCCTTAAGGAGAAG
c. TGGACATCATGACAGCAACCCTTAAGGAG AAG
d. GATATACATATGCACCATCACCACCATCA TGCCATGGCAACCCTTAAGGAGAAG
Tipranavir Author
wrote...
#2
3 years ago Edited: 3 years ago, Tipranavir
Quote from: bio_man (3 years ago)
Hi Tipranavir

Looking at your first question about forward primers, would you happen to have the start sequence for the mouse? Reason I ask is because you're expected to choose the option that complements the anti-sense strand of the mouse. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).

Quote
You want to clone the CDNA for mouse LDHB (NM_008492.3) into pET11-H5alpha. Which of the DNA sequences below could be used as forward primer for this purpose?

a. CCATGGCAACCCTTAAGGAGAAG
b. TGGACACCATGGCAACCCTTAAGGAGAAG
c. TGGACATCATGACAGCAACCCTTAAGGAG AAG
d. GATATACATATGCACCATCACCACCATCA TGCCATGGCAACCCTTAAGGAGAAG

Hey, thank you so much for doing this!

I believe we're supposed to use this thing called "BLAST" to get the sequence of this.  https://blast.ncbi.nlm.nih.gov/Blast.cgi  The question gives this "NM_008492.3" identifier, which I believe corresponds to cDNA for mouse LDHB.  I gave the BLAST tool a try (chose "Nucleotide BLAST"), entering "NM_008492.3" into the box that's labelled "Enter accession number(s), gi(s), or FASTA sequence(s)", then clicked "BLAST" at the bottom without changing any options (there are so many, it's daunting).

https://imgur.com/L60HL1G

Choosing the first result, I got the following
https://imgur.com/XpEEHpk

Am I doing this right?
wrote...
#3
Educator
3 years ago
Yes, that's how its done. Did you find any matches?

Mind also sending me the URL for your BLAST search?
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