Jump to content

  • Log in with Facebook Log in with Twitter Log In with Google      Sign In   
  • Create Account

boa

Member Since 01 Oct 2012
Offline Last Active Oct 01 2012 08:10 PM
-----

Topics I've Started

recognition sites and cloning?

01 October 2012 - 08:12 PM

Hello all, I have absolutely no clue how to start this problem and I would like to have some cues or just something to chew on...thank you!!
I know what a plasmid is , I know what sticky ends (cohesive extremities) are but I dont know how the multiple cloning sites in this plasmid are located so im a bit lost!

Question: U must clone a DNA fragment having Sau3A sticky ends.
The plasmid to be used for the cloning experiment has the following MCS
EcoR1, BamH1, Xba1, HindIII

*Describe how you would do the cloning experiment.

Restriction enzyme recognition sites
EcoR1 = GAATTC (the enzyme cuts after the G)
BamH1 = GGATTC (the enzyme cust after the first G)
Xba1 = TCTAGA (the enzyme cuts after the first T)
HindIII = AAGCTT (the enzyme cuts after the first A)
Sau3A = GATC (the enzyme cuts before the G)

question about PCR and cloning

01 October 2012 - 08:11 PM

I have no clue where to start but I think it is safe to say that my forward primer could be TAC and my reverse primer TTA (complementary sequences of the start and stop codon) but I dont know what to do after...the problem seems very overwhelming to me! I would like someone to provide me some hints to tackle this!
Thanks everyone!Thank you for your help!

Question :
Write the sequence of two oligonucleotides that will allow you to clone the coding region of gene x
in the vector pQE60 using PCR . The coding sequence must be in frame with the ATG of the vector. The histidine tag must be present
at the C-terminus of your recombinant protein. The oligos must be as short as possible but must
hybridize with 20 nucleotides of the template sTrand. The start and stop codons of gene X are
underlined.

Coding sequence of gene X
GTCGATCAAT ATGGAACATG TTTACTCCAA ACCACCGCAC ACCAATTATG
GAAACCAAGC CGGAAAAGAA TTCCGGTGGA GAGCGAAAAA AAAGGATTCC
GAATCGTGAA CTGCCAAAAA CATTTTGAAG CCAACGATTC CGACGTCATC
CTCGCCACCC TAGCTAAATC AGGCACCACT TGGTTAAAAG CTCTTCTCTT
TGCTCTCATT CACCGACACA AGTTCCCAGT TTCTGGCAAG CATCCTCTTC
TGAAACAGCA GTAGCAGCGT TTAAAGGGAA GTTTATT

Oligo #1 5' ________________________________________…
Oligo #2 5' ________________________________________…

Nco1 = CCATGG BamH1 = GGATCC BglII = AGATCT HindIII = AAGCTT

pQE60 (RBS)CCATGGGAGGATCCAGATCT(blackbox) TAAGCTTCCGCATAATTAGCTGAG
Additional Details
The start and stop codons of gene X are the first ATG and the last TAA

underlined in vector pqe60: the first ATG

Home - About - Terms of Service - Privacy - Contact Us

©1999-2012 Protocol Online, All rights reserved.