S-STEM Scholar Leilani Boren's Blog

 Introduction:  The previous week revealed that the Hilgarth protocol could be disregarded going forward and the Anna protocol could and should be utilized for the greatest chance of success of the assembly of the three fragment plasmid. This plasmid, as a reminder of the eventual goal of this project, contains an antibiotic resistance gene that will hopefully be expressed after homologous recombination of this linear plasmid directly into the genome of competent D. radiodurans cells. Individual fragments were gel excised and cleaned in order to undergo overlap PCR again with left and tet, while the "optimal" temperature of overlap being 36 degrees Celsius suggested by Snapgene is simultaneously verified. Once left and tet overlap can be accomplished, then an attempt can be made for the full three part product needed for transformation.  Methods:  All gels were 1.2% agarose with 1x TAE solution unless otherwise stated, with a 1 kb gene ruler ladder.  The gel ex...

Introduction: Previous weeks have resulted in confusion surrounding multiple aspects of the overlap method for a three piece linear plasmid construction. Two protocols have been utilized thus far, Hilgarth being the primary focus for the most recent weeks given their undisputed success of overlap and minimalization of ghost banding due to techniques to increase specific binding and primer specificity for overlap and amplification steps. These techniques include using diluted primers, adjusting the template DNA loaded into PCR reaction, and utilizing touchdown PCR. That being sad, at the beginning of the semester, fragment isolations from D. radiodurans template DNA for the left and right, and isolation of tetr/a gene from an E. coli plasmid using standard PCR had been successful with minimal ghost banding, and overlap of left and tetr/a had been accomplished, thought not efficiently, with the Anna protocol. This week an attempt to use gel excised fragments of left and tetr/a assemble...

 Introduction:  Last week provided insight to how we have been misunderstanding the Hilgarth protocol for overlap assembly of the three part fragment that will hopefully be transformed into D. radiodurans. It was realized that the correct amount of DNA may not have been loaded for the initial overlap reaction, which excludes primers. Both protocols, the Anna and the Hilgarth, stress equimolar inputs of fragments, as well as correct temperature conditions. Both of these factors have not been done correctly for our fragments, as the Hilgarth paper has fragments with homologous regions with an ideal temperature of 65 degrees Celsius, and our fragments are nearly half at 37 degrees Celsius. Therefore, following the Hilgarth protocol identically to the paper will very likely not yield results for our three part assembly. This week will focus on the Hilgarth protocol and establishing whether or not overlap of the left and tet assembled fragment with the right fragment is possible, a...

 Introduction:  Previous work in overlap ligation has revealed problems amplifying the assembled left and tetr/a fragment in order to complete a full three part plasmid. This was deemed necessary at the time to acquire enough stock fragment to undergo multiple attempts at overlap ligation introducing the right fragment, but it was decided to go forward with what assembled fragment remained and attempt to attach the right fragment anyways. The PCR experiments this week evaluated two primary methods for overlap ligation, the Anna protocol (Behle 2019) and the Hilgarth method (Hilgarth et al 2020). The Anna protocol has been used thus far, including the successful overlap of left and tetra fragments. The Hilgarth protocol was introduced due to other successes in the lab in which assembly was achieved, with very high specificity of products (very little ghost banding). The restriction enzyme ligation team isolated all three fragments the previous week, and went forward by attempti...

 Introduction:  The previous weeks' work resulted in an overlap assembled fragment with left and tetr/a that was undergoing very nonspecific binding as seen in agarose gels with significant smearing. The restriction enzyme ligation team digested and phosphorylated all three fragments simultaneously, and amplified the predicted product. This week focused on figuring out how to achieve more specific amplification of the left and tetr/a assembled overlap fragment, and evaluating restriction enzyme results and preparing new fragments.   Methods:  All gels were run on 1.2% agarose unless otherwise stated, with a 1 kb ladder.  Overlap:  Two microliters of final PCR (PCR product of assembled left and tetr/a fragments) product were directly amplified with no PCR cleanup in a 50 ul reaction on a heat gradient from 68 to 58 degrees Celsius annealing temperature for 30 cycles. A gel was prepared and ran on this final PCR product for an extended length of time at ...