1. You will estimate the quantity of DNA present in the pUC18 DNA prep by gel ba
ID: 193447 • Letter: 1
Question
1. You will estimate the quantity of DNA present in the pUC18 DNA prep by gel band brightness. Compare your linear plasmid band in the HindIII digest lane to the -HindIII marker. If, for example, your linearized plasmid is half as bright as the 4, 361 bp band of the marker, then your approximate DNA quantity is 90/2 or 45 ng. You loaded 10 l of the HindIII digest onto the gel so your original concentration of DNA in the digest is 45 ng/10 l or 4.5 ng/l. You put 10 l of DNA in your digest. All together then, you have 4.5 ng/l x 10 l or 45 ng of DNA in the digest.
I am comparing my linear Hind III digest plasmid to Fragment ____ of the -HindIII digest.
The amount of DNA in the linearized plasmid is ________ and I reach this number because_________________________________________________________________.
The original concentration (g/l) of pUC18 plasmid DNA in the HindIII digest is __________________.
The total amount of pUC18 plasmid DNA in the HindIII digest is ______________g.
The pBR322-BstNI digest cannot be used for quantitation but can be used to estimate fragment size. You may find this marker useful in analyzing your data (but quantitate the DNA using the lambda-HindIII digest marker).
Tube I: no enzyme
Tube II: 1 l HindIII
Tube III: 1 l HindIII, 1 l ScaI
Tube IV: 1 l BglI, 1 l ScaI
This is an example of how to answer this question
I am comparing my linear HindII digest plasmid to Fragment 4 of the -HindIII digest. The amount of DNA in the linearized plasmid is 45 ng and I reach this number because the assume band looks about half as bright compared to Fragment 4 of the -HindIII digest. The original concentration of pUC18 plasmid in the HindIII is 45 ng/10 l or 4.5 ng/l. The total amount of pUC18 plasmid DNA in the HindIII digest is 4.5 ng (because I added 10 l of DNA in the digest, so 4.5 ng/l x 10l = 45ng). Too much ethidium bromide was added into the solution, which cause about half of the gel to fluoresce. Therefore, I have to guess the sizes of the marker fragments, which means that this estimation is not very accurat
23,130 Base Pairs 23,130 9.416 6,557 4,361 2,322 2,027 564 125 Fraqment DNA Mass 477 n 194 n 135 n 90 n 48 n 42 n 12 n 3 n - 94161 5,257 3 3614 5 6 7 2,322 8 2027 -HindIII Digest (Marker) //www.neb.com/nebecomm ts intl 3012.aExplanation / Answer
I am comparing my linear HindII digest plasmid to Fragment 4 of the -HindIII digest. The amount of DNA in the linearized plasmid is 45 ng and I reach this number because the assume band looks about half as bright compared to Fragment 4 of the -HindIII digest. The original concentration of pUC18 plasmid in the HindIII is 0.0045 g/l (4.5 ng X 1000/1000/l) The total amount of pUC18 plasmid DNA in the HindIII digest is 0.45 g (because I added 10 l of DNA in the digest, so 4.5 ng/l x 10l = 45X 1000 / 1000 ng).
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.