2. Bio Design
20th of September - George Church (Harvard)

 

DNA modification is fundamental to design in synthetic biology. For this week's assignment, you will learn to read DNA sequence files and see physical changes you will make to DNA molecules.
 

Lab Task:

 

Verify restriction digest of a circular DNA plasmid by gel electrophoresis. Compare your experimental result with what you would predict from the plasmid map.

 

​

​

​

​

​

​

​

​

​

​

​

​

​

 

 

 

 

Plasmid pUC19 contains the restriction sites PvuII, these sites are in positions 52 and 374.
When we use the enzyme PvuII, it will cut these sites, which will produce fragments of 322pb and 2364pb.

​

​

​

​

Work Flow

1.In two separate tubes sized appropriately for your 37C source, mix the followingItem Vol to RxpUC19 ( 1ug / ul) - 2 ulPvuII-HF (20 Units / ul) - 1 ulCutsmart Buffer (10x) - 3 ulDeionized Water - 24 ul

2.We incubated one reaction mix at 37C (line 01), ~4C (line2) and control for 1 hr.

​

3.We casted a 1% agarose TAE gel 

-Add 1 g agarose to 100 mL 1x TAE (10 mL 10x TAE and 90 mL water) and swirl
-Microwave until agarose is fully dissolved (2-3 min) and before solution boils over
-Mix in 10000x DNA gel stain (10 uL for every 100 mL gel)
-Cast the gel in a tray with a well-forming comb inserted and wait ~1 hr to set (solidify)

4.We loaded the gel wells with completed digests 

-Orient solid gel so wells line up on the negative electrode side of the electrophoresis cell ("box")
-Fill gel box with 1x TAE until the gel is submerged and remove comb
-Pipette DNA ladder into the first well.

5.The mix ran for ~1 hr at 120V.

​

6.Image DNA separation through an orange filter over a blue-LED transilluminator.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusion: we found the expected results according to the cuts of theoretical restriction sites in lines 1 and 2. The enzyme works best at temperature 37C, but also showed that it can perform cuts at temperature 4C.

​

Gel electrophoresis is the methods more common but more important to molecular biology. Can be used for a range of purposes, for example:

To check a PCR reaction.

To Separation of restricted genomic DNA prior to Southern transfer.

To check integrity of genomic DNA

To test for genes associated with a particular disease.

To DNA profiling for taxonomy studies to distinguish different species

To DNA fingerprinting to investigate crime scenes

To the study of evolutionary relationships by analyzing genetic similarity among populations or species

​

Theory Task: Choose a DNA sequence that interests you from nature or a source like the iGEM part registry. Provide a detailed workflow for isolating or acquiring a molecule with this specific sequence and an assay that confirms part of its content.

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

​

To obtain the myostatin gene, we obtain a blood sample, then RNA is extracted, then a cDNA synthesis is made, with specific oligos, it is amplified and the master mix is amplified in the thermocycler fragment. Finally it is revealed in an electrophoresis if the fragment is correct. Subsequently, a DNA purification can be performed and the fragment sequenced.

​

​