Molecular Cloning write-up #1 1.Using the picture of the gel provided to you, es

Question

Molecular Cloning write-up #1 1.Using the picture of the gel provided to you, estimate of the number of restriction fragments created by each digest of the chromosome by counting the number of bands in each lane. HindIII digest of the plasmid: BamHI digest of the chromosome EcoRI digest of the chromosome HindllI digest of the chromosome: pUc19 Chrom Chrom Chrom tmn Creom 2 Estimate the size of each DNA fragment identified in step 1 a) Make a standard curve relating the distance migrated during electrophoresis to their size in bp (base pairs) using the molecular weight marker DNA lane (the DNA ladder). Start from the top of the gel (at the wells) and measure the distance traveled by each band in the ladder (measure to each band). Plot those distances on the semilog graph page supplied (in cm) on the linear X axis and record the size (in bp) on the logarithmic Y axis. See the handout on your bench for an example graph (but do your ownl). Then use the standard curve to answer the next question(s) b) Use the standard curve to estimate the size of the restriction fragments you identified as bands in step HindllI digest of the the plasmid: BamHI digest of the chromosome: EcoRI digest of the chromosome: HindIll digest of the chromosome 3. Estimate the total size (length in kb) of the chromosome. Add up the sizes of restriction fragments (from #2) to determine the length of the intact DNA molecule before restriction digestion. Each digest will give you a estimate of total size. Even though the same chromosome was cut with different restriction enzymes, it is possible that all three estimates will not agree (you will discuss this in N4) BamHl digest of the chromosome: EcoRI digest of the chromosome: HindI digest of the chromosome:

Explanation / Answer

1. In case of HindIII digest of plasmid, no digestion has occured. In this case a circular plasmid have been used and if there would have been a single digestion (which might have occured as only one band is present), then there would have been a formation of a linear DNA. Now, if the DNA is linear then the movement occured through the gel is usually slow compared to the circular one. In all the other cases, since chromosomal DNA has been used, the form of the DNA is linear. So, the number of restriction digestion would be one less than the number of bands present on the gel.

BamH1 : 5 fargments

EcoR1 : 6 fragments

HindIII : 6 fragments

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