Continuation of Overlap and RE Ligation PCR Techniques for Linear Plasmid Construction

 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 10 mAMPs in order to get increased accurate visualization of the suspected assembled fragment to try to explain the smearing seen in reactions after this sample. A reaction mix with just PCR water instead of DNA template was also ran to evaluate possible  contamination of those stocks. 

Left and tetr/a fragments were re-isolated utilizing a comparable technique using a 50 ul reaction with a decreased amount of template DNA and primers being added to the reaction. 10 ng of template and 1.5 ul of 10 uM primers were loaded for each fragment, and ran on touchdown PCR with a temperature range starting at 67 degrees Celsius and decreasing 0.5 a degree each cycle. 

The left and tetr/a assembled fragment was attempted to overlap with the right fragment, loading 100ng of each of the two fragments into a 24  ul reaction at 37 degrees Celsius for annealing temperature as the median between the optimal of the two homologous regions of each fragment. 

A gel was run for each sample that could be visualized with sufficient DNA concentration. 

Restriction Enzyme Ligation:

A gel was made for evaluating results of attempted ligation of all three fragments, with an extended run time for optimal clarity in results. 

Each fragment was isolated from template DNA or plasmid in 50 ul reactions at 60 degreese Celsius annealing tmperature, with 2 ul of DNA loaded for each. 

Results: 

In Figure 1 there is significant ghost banding primarily below the desired fragment, but no bands besides suspected primers in the lane with just PCR water. In Fig. 2 the tetr/a and right fragment overlap did not show bands, but appears to have stayed in the well. The restriction enzyme ligation was unsuccessful in both lanes with large amounts of smearing. In Fig. 3 the heat gradient trying to replicate the left and tetr/a assembled fragment showed no real change between any temperatures tested, they were all smears rather than bands we were looking for. In Fig. 4 the left fragment was isolated successfully with touchdown PCR but the tetr/a fragment was smeared. In Fig. 5 the three restriction enzyme fragments showed little to no ghost banding and seems to have produced high levels of concentration. 


1:From left to right- final PCR product and PCR water reaction with primers. 
 
2:From left to right- Tetr/a and right fragment overlap attempt, restriction enzyme ligation product with standard PCR for amplification, restriction enzyme ligation product with elongated PCR.  

3:Heat gradient of left and tetr/a fragment assembled and amplified, left to right from 68 to 58 degrees Celsius. 


4:Overlap left fragment re-isolated with touchdown PCR and tetr/a fragment with touchdown PCR, each with 10 ng loading template. 

5:Restriction enzyme fragments, left, tetr/a, and right. 



Discussion: 

Restriction enzyme ligation may have been unsuccessful on the first try, but the newly isolated fragments are exceptionally specific and concentrated, particularly the tetr/a fragment. They can now go forward digesting and phosphorylating these samples after an additional amplification is down to ensure plenty of stock fragment is acquired. Needing such a high mass of each fragment, we are trying to avoid having to isolate fragments again in the near future. Overlap PCR results lead us to not trust the left and tetr/a fragment seen last week, although we have seen it again this week. The amount of nonspecific priming after amplifying a second time is concerning and wants to be avoided at all costs. The new protocol with less loading template and using touchdown PCR is meant to cut down on this nonspecific priming, as well as in the future diluting primer concentrations to 1 uM in order to encourage more specificity post overlap of fragments in the subsequent amplification step. The tetr/a fragment that became a smear on the gel post isolation is likely due to poor plasmid template quality, possibly heavily contaminated with genomic DNA, which would lead to these skewed results.      

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