Continuation of Linear Plasmid Construction by Restriction Ligation and Overlap Techniques

Introduction:

    From the previous week, two of three fragments needed for linear plasmid construction in order to successfully transform Deinococcus radiodruans had been visualized on an agarose gel for overlap PCR. This week, it was decided to also pursue a second route in plasmid construction, restriction enzyme ligation. By dividing into two groups the team will be able to separately test overlap PCR and RE ligation techniques and evaluate differences between the two methods in terms of time, resources, 0=d obstacles. The fragments will be independently isolated with RE ligation specific primers to move forward in that direction. 

Methods:

    Deinococcus radiodurans was isolated from Mueller-Hinton broth and DNA extraction was completed using the Ultraclean DNeasy extraction kit for template PCR DNA. E. coli was also inoculated in LB broth and grown statically at 37 degrees Celsius to acquire more template plasmid DNA using a Monarch plasmid extraction kit. Both samples were tested on the Nanodrop for concentration and purity. 

  Overlap PCR

   Samples one through eight from the heat gradient PCR testing of tetr/a  were compounded and cleaned with BioLabs Monarch PCR Cleanup kit. The eluted sample was tested on the Nanodrop and diluted to an approximate concentration of 100 ng/ul. The right and left fragments were run on a 58  to 68 degrees Celsius heat gradient and the 58 and 60 degree samples were combined and cleaned in the same process as the tetr/a fragment. Agarose gels (1%) were ran for each fragment before and after cleanup to ensure molecular weight and primer specificity. 

Restriction Enzyme Ligation

    The tetr/a, left, and right fragments were isolated by PCR with appropriate primers at 60 degrees Celsius annealing temperature. A 1% gel was ran with a 1kb ladder to validate fragment identity and primer specificity. Post gel the primers and each PCR product were tested on the Nanodrop to confirm DNA concentrations. 

Results:

The right fragment proved to be successful for overlap PCR once the right primers were used. The tetr/a fragment(shown below) was confirmed as well as the left and rigth after cleanup, although bands were less obvious which indicated less concentration. The RE fragments were unsuccessful as seen on the gel below, leading to the testing of the five PCR products and one of the primers, which did in fact show significant levels of DNA and primer concentration which should have been seen on the gel. The template DNA and plasmid isolation were minimally successful for their intended use in the project going forward. 

Agarose gel with tetr/a fragment for overlap PCR post cleanup kit. 

1kb ladder with three PCR products for RE ligation, tetr/a, left, and right fragments. 

Sample	Concentration Ng/ul
D. rad genomic DNA	86.0
E. coli plasmid isolation	30.0
RE tetr/a fragment	150.1
RE left fragment	89.8
RE left fragment	124.2
RE right fragment	69.4
RE right fragment	181.0
RE primer P1	94.2

Table of all Nanodrop readings taken for template DNA and RE PCR products post gel results, as well as one sdDNA reading for primer confirmation. 

Discussion: 

    The overlap PCR fragments seem to be validated and ready to move forward with overlap attempts for ligation, starting with two fragments at a time. The RE fragments were mysteriously missing from the gel despite Nanodrop readings that showed substantial levels of concentration, but this is likely contributed to potentially bad Mastermix, due to being left at room temperature for too long. These fragments will be redone next week and if still not successful, the annealing temperature will be further evaluated to see if an increase or decrease is needed for higher primer efficiency. The plasmid extraction will also likely be redone to produce a higher concentration, since 30 ng/ul is doable but not as favorable as we had hoped. It is thought that using a culture with a higher OD600 value will help yield better results.