Polymerase chain reaction-based gene removal from plasmids.

This data article contains supplementary figures and methods to the research article entitled, "Multiplex gene removal by two-step polymerase chain reactions" (Krishnamurthy et al., Anal. Biochem., 2015, doi:http://dx.doi.org/10.1016/j.ab.2015.03.033), which presents a restriction-enzyme free method to remove multiple DNA segments from plasmids. Restriction-free cloning methods have dramatically improved the flexibility and speed of genetic manipulation compared to conventional assays based on restriction enzyme digestion (Lale and Valla, 2014. DNA Cloning and Assembly Methods, vol. 1116). Here, we show the basic scheme and characterize the success rate for single and multiplex gene removal from plasmids. In addition, we optimize experimental conditions, including the amount of template, multiple primers mixing, and buffers for DpnI treatment, used in the one-pot reaction for multiplex gene removal.


Specifications
Value of the data The data provide a method and systematic characterization for removing one gene from plasmids based on inverted PCR and blunt-end ligation.
The data provide the scheme and characterize the success rate for PCR-based multiplex gene removal.
The data provide the optimization of methods, including the amount of parent template, multiple primers mixing, and buffer conditions for DpnI digestion, to achieve pure products with multiple genes removed from plasmids based on two rounds of PCR.
1. Data, experimental design, materials and methods

Primer design
Single gene removal was achieved by inverted PCR with two 5'-phosphorylated primers, followed by blunt-end ligation. Multiplex gene removal was achieved by two-step PCR. In such a setting, for each gene segment to be removed, three non-phosphorylated primers were sufficient: two for the first round of PCR and one for the second round of PCR. For the first round of PCR, shared by both single and multiplex gene removal, we designed the primers so that the sense primer overlapped with the downstream sequence of the vector and the antisense primer overlapped with the reversecomplementary upstream sequence of the vector. We varied the length of both primers between 18 and 22 bases in lengths so that their annealing temperatures were within 4 1C of each other. If bluntend ligation was used, the third primer was not needed. This inverted PCR-ligation method worked well for removing one gene from plasmids with sizes up to 9.6 kb ( Fig. 1). For the second round of PCR, each single-stranded oligo was 40 bases in length and had a 20-nt complementarity with the two fragments to be connected [3,4]. Fig. 2 shows one possible scenario for three gene removal based on a specific single-stranded oligo, FA.

Two-step PCR
To linearize the vector in round-1 PCR, 10 ml Phusion polymerase 2 Â master mix (NEB) was mixed with 1 ml of sense and antisense primers (10 mM), 1 ml template (10 pg/ml), and 7 ml of water.
In each reaction cycle, the reaction mixture was denatured at 98 1C for 15 s, annealed for 15 s, and extended at 72 1C. The yield of PCR products did not change significantly when the amount of template was adjusted from 1 pg to 5 ng (Fig. 3). When multiple primer pairs were used, the lowest annealing temperature of all primers was used. Mixing three pairs of primers did not degrade the quality of linear products compared to separated primer pairs (Fig. 4). The extension time depended on the longest linear product with 1 kb/min extension rate (e.g. 2 min for a 2 kb linear product).
After the reaction, the PCR product was mixed with 2 ml DpnI (10 U/ml) and incubated at 37 1C for 1 h, followed by PCR clean-up. No buffer exchange was required for DpnI treatment, because enzymatic activity of DpnI did not degrade in the PCR master mix of Phusion DNA polymerase (Fig. 5). The final concentration of the fragments was measured by NanoDrop. A typical concentration of the products ranged from 30 to 50 ng/ml. The linear fragments were then circularized by the second round of PCR extension. To set up the round-2 PCR, 250 ng of linear fragments were mixed with 10 ml Phusion polymerase 2 Â master mix (NEB), 1 ml of ss-oligos (20 mM each), and water to make a 20 ml reaction mix. In each reaction cycle, the reaction mixture

Transformation
DH5α (provided by Dr. Sandra McMasters in the cell media facility in UIUC) competent cells were used for transformation. Briefly, 30 ml thawed competent cells were mixed with 5 ml products of bluntend ligation reactions or two-step PCR and incubated on ice for 30 min. Cells were then incubated at 42 1C for 45 s and transferred back to ice and incubated for another 2 min. Cells were then incubated in 1 ml Luria-Bertani (LB) media at 37 1C with vigorous shaking for 1 h. Two hundred and fifty microliters of cell culture were evenly spread onto agar plates and incubated at 37 1C overnight.

Blue/white colony screening assay
To pre-made LB agar plates, one hundred and twenty microliters of X-gal stock solution (20 mg/ml stock in Dimethylformamide) were added and spread evenly using glass spreaders at room temperature. The plates were incubated at 37 1C for at least 30 min to dry. The recovered competent cells were then plated and the plates incubated at 37 1C overnight [5]. This assay worked for plasmids with sizes ranging from 4.0 kb to 9.6 kb. After removal of one gene from the vector (the sizes of the genes were indicated in the parentheses), the circular plasmids were amplified. All plasmids (before and after gene removal) were then digested by BamHI, which was a unique cutting site for all plasmids. Successful removal of genes was observed in all four plasmids.

Colony PCR reaction
For each colony PCR screening reaction, eight colonies were randomly picked from the agar plate. Each colony was grown in 4 ml LB media in a 14 ml cell culture tube with appropriate antibiotics at 37 1C for 4 h with vigorous shaking, which gave a slightly turbid culture if the colony was successfully transformed. One milliliter of cell culture was then taken from each cell culture tube and transferred to a microcentrifuge tube. The cultures were spun down at 13,000 rpm for 1 min in a mini-centrifuge (Eppendorf). Supernatants were discarded and each cell pellet was resuspended with 50 ml sterile water. Cells were then lysed at 100 1C in a dry heat bath for 5 min and cooled on ice for 2 min. The cell lysates were spun again at 13,000 rpm for 1 min. Two microliters of clear supernatants were used as templates for the following colony PCR (Table 2). After the reaction, products were loaded onto a 1% Fig. 3. Dose-dependence of the yield of PCR products on the amount of parent template. In a three-gene removal case (u85, f1, and kanR), 1 pg was sufficient to generate enough products. Lane 1: DNA ladder; Lane 2: 10 ng template alone; Lane 3-7: PCR products with 1 pg to 5 ng of template. Fig. 4. Mixing three pairs of primers did not degrade the quality of linear products compared to separate primer pairs. Ta: annealing temperature. Lane 1: DNA ladder, Lane 2: DNA template alone, Lane 3-5: PCR products with primer pair 1, 2, and 3 in three separate reaction, Lane 6: PCR products with all three pairs of primers mixed in one reaction. agarose gel and run at 90 V for 30 min before images were taken on a blue transilluminator. For the experiment of three gene removal, 8 out of 8 randomly selected colonies produced plasmids with the correct size (Fig. 6). Fig. 6. Colony PCR showed that products from 8randomly selected colonies had all three gene segments (u85, f1, and kanR) removed from the plasmid pCRII-U85. Lane 1: DNA ladder, Lane 2: products of full-length pCRII-U85. Lane 3-?10: products of eight randomly selected colonies.