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

 Introduction:

In the previous weeks, the fragments of left, tetr/a, and right have been isolated for overlap PCR and identical fragments for RE ligation were beginning to be processed. This week consisted of successfully isolating RE fragments, as well as cleaning all fragments for both methods. Then digestion was started for RE fragments while overlap binding was attempted for the left and tetr/a fragments with PCR. 

Methods: 

All amplifying PCR reactions with a 20 ul total volume consisted of 16 ul of Mastermix, 2 ul of DNA template, and 1 ul of each primer. Alll reactions with a volume of 50 ul were done with 43 ul of Mastermix, 2 ul of DNA template, and 2.5 ul of primers. Gels were consistently 1x TAE 1% agarose with a 5 ul of sample loaded for each lane.  

The RE fragments were isolated in 20 ul PCR reactions with freshly made Mastermix to correct the lack of results from the previous week's gels. Each sample was processed in the standard Monarch PCR cleanup kit and eluted in increments of 10 ul, testing concentration on a Nanodrop each time, and combined once sufficient DNA was achieved. The aliquots were combined for a total of 16 ul per fragment. Approximately 1 ug of each was loaded into a single digestion reaction so phosphorylation and ligation could be attempted next week. 

Overlap fragments were purified with the standard Monarch PCR cleanup kit in an identical process, each resulting in 16 ul of total volume. Overlap ligation of the left and tetr/a fragments was attempted with 50 ng of total mass input into the PCR reaction in a rough 1:1 equimolar ratio. This reaction was devoid of primers due to the fact amplification would be sought after in the following PCR reaction. 5 ul of 5x Q5 buffer, 5 ul of 5x high GC buffer, 0.5 ul of dNTPs, and 0.5 ul of Q5 polymerase was utilized, and the remaining volume to reach 24 ul of total solution was reached with PCR water. This reaction was run on 15 cycles with an ideal homologous region annealing temperature of 36 degrees Celsius. This reaction was stored and 2 ul used for the secondary amplification step, which was done at 50 ul of total volume with primers 1 and 6. A gel was done to confirm assembly and amplification. The desired fragment was isolated be gel excision and purified with the ThermoFisher GeneJET gel extraction kit. Two 50 ul volume PCR reactions were run to attempt to isolate and amplify the desired fragment further, one with 2 ul of the sample derived from gel extraction, and the other 2 ul of template DNA from the previous PCR reaction directly (only 10 ul) of the total volume). Each sample was cleaned with standard cleanup kit prior to PCR at annealing temperature of 60 degrees Celsius. Samples were also run on a gel and measured on the Nanodrop to evaluate primer specificity and approximate concentration. 

Results:

The RE fragments were all confirmed, but tetr/a is pictured below at 700 bp. The data in table 1 overviews all samples tested at different stages, most being sufficiently cleaned after PCR cleanup with the exception of post overlap assembly fragments. The second gel pictured includes the top band being 1700 bp desired fragment, as well as two fragments close to 1000 and 700 that is assumed to be amplified due to leftover primers from previous reactions. Final gel shows the cleaned products from gel 2, unsuccessful with no desired bands and many smears. 

RE ligation tetr/a fragment confirmed. 

Sample	Concentration ng/ul	260/280	260/230
Overlap
Left	95.0	1.76	1.90
Right	120.0	1.85	2.16
Post Gel Extraction
Left and Tetr/a assembled	6.7	1.60	0.06
Post Cleanup
Left and Tetr/a gel extraction	59.9	2.08	-0.41
Left and Tetr/a from PCR	1.80	1.24	-0.01
RE Ligation
Left	118.0	1.82	2.00
Right	150.0	1.80	1.90
Tetr/a	120.0	1.94	2.50

Gel post overlap ligation using a 1kb ladder, the top band being approximately 1700 bp. 

Overlap assembly product left lane post gel and the right lane post PCR directly. 1 kb ladder

Discussion: 

With the digestion completed on the RE fragments it is yet to be seen after phosphorylation and attempted ligation if these fragments will need to be isolated again, and if they are going to be successfully assembled. Overlap PCR resulted in a fragment deemed to be the desired 1700 base pairs, but subsequent cleaning and isolation was obviously unsuccessful due to the nature of the most recent gel. It is a mystery as to why this gel did not show a 1700 bp band, and exhibited such a broad smear. This leads us to reconsider how we are purifying this sample post assembly amplification, and evaluate if there were any human mistakes during these purification stages. These samples did exhibit strange Nanodrop readings, and although we proceeded anyways, it is possible we could have predicted this outcome prior to the reaction. It will be essential to isolate the left fragment more efficiently and still have the desired band prior to introducing the right fragment for the entire linear plasmid to be complete.