4 replies to this topic

[molecularralph]

For the double-stranded DNA fragment below, give the sequences you would expect for probes labelled by a) random priming, b ) end-filling of EcoRI sites or c) end-labelling. Circle all nucleotides in the probes that will have incorporated radioactive phosphorus. 

 

 

5′-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′

3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAGGTTAT-5′

 

a) Probe made by random priming with the hexameric primers TGGAAT AND GATTAG:

 

 

 

 

b ) Probe made by end-filling of EcoRI sites:

 

 

 

 

c) Probe made by end-labelling:

 

Obvious homework... moved. Bob

Edited by bob1, 24 October 2014 - 01:27 AM.

[bob1]

Some input from you would be good... help yourself by showing some thought.

[molecularralph]

This is what i have so far. I am having trouble with this subject. If someone can just guide me it will be helpful. thank you

 

 

a) Probe made by random priming with the hexameric primers TGGAAT AND GATTAG:

 

5′-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′

3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAGGTTAT-5′

 

 

 

b ) Probe made by end-filling of EcoRI sites:

 

5′-GAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTC-3′

3′-CTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAG-5′

 

 

 

c) Probe made by end-labelling:

 

5′P³²-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′

           ê

 

5′-      TTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′

3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAA      -5′

 

                  ê

5′-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′

3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAGGTTAT-5′

Edited by molecularralph, 24 October 2014 - 11:41 AM.

[molecularralph]

Some input from you would be good... help yourself by showing some thought.

sooo?? nothing

[bob1]

This is what i have so far. I am having trouble with this subject. If someone can just guide me it will be helpful. thank you
 
 
a) Probe made by random priming with the hexameric primers TGGAAT AND GATTAG:
 
5′-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′
3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAGGTTAT-5′

usually when making these sorts of things, only one radiolabeled nucleotide is included, so only some bases will be labelled. For this one the probes will be the nucleotides between the primers...
 

b ) Probe made by end-filling of EcoRI sites:
 
5′-GAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTC-3′
3′-CTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAG-5′

 EcoRI cuts at the space: G AATTC leaving an overhang, end filling will fill in the overhang.
 

c) Probe made by end-labelling:
 
5′P³²-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′
           ê
 
5′-      TTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′
3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAA      -5′
 
                  ê
5′-GCAGAATTCGATTAGCAATGGACTGGGCCAAATTGCGAATTCCAATA-3′
3′-CGTCTTAAGCTAATCGTTACCTGACCCGGTTTAACGCTTAAGGTTAT-5′

End labeling will substitute the only the end nucleotide, but it depends on which end you are doing the subsititution on (5' or 3' end labeling?).