Column-Based Nucleic Acid Extraction and Purification

Poh and Gan, J Bioproces Biotechniq 2014, 4:3 http://dx.doi.org/10.4172/2155-9821.1000157 Bioprocessing & Biotechniques Research Article Open Access The Determination of Factors Involved in Column-Based Nucleic Acid Extraction and Purification Jun-Jie Poh1,3 and Samuel Ken-En Gan1,2,3* 1 Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), 138671, Singapore 2 p53 Laboratory, Agency for Science, Technology, and Research (A*STAR), 138648, Singapore 3 Quintech Life Sciences Pte Ltd, 619933, Singapore Abstract DNA extraction methods such as plasmid minipreps, gel, and PCR purifications, are indispensable techniques for genetic manipulations. There are numerous factors that contribute to the efficiency of these processes, which determine the success of complex downstream molecular analytics and diagnostic tests. To study and optimize these factors, we compared our own proprietary buffers to commercially available column-based kits, utilizing their spin columns and protocols. Through systematic substitution of the buffers in the kits with our own proprietary buffers, we selected the highest DNA yielding buffer recipes. Further analysis of the differences between the buffers showed that high concentrations and presence of certain chaotropic agents and cations are necessary for good plasmid miniprep, gel extraction, and PCR purification kits. Keywords: DNA extraction; Gel extraction; PCR purification; Based on the same principle for plasmid DNA extraction, spin Column-based purifications columns had also been used for gel extractions and polymerase chain reaction (PCR) purifications. While these developments have Abbreviations: E. coli: Escherichia coli; SDS: Sodium Dodecyl contributed greatly to biomedical research, major developments of Sulfate; TE: Tris-EDTA; OPT: Optimized; HM: Home-Made these kits are generally largely focussed on membrane material science. To complement this, we have decided to investigate whether the Introduction manipulation of chemicals in associated buffers will increase the yields The extraction and purification of nucleic acids are commonly used that would enable researchers to tweak their existing commercial kits techniques to isolate genetic material from tissues, bacteria, plants, and for improved yields. viruses for important analytical, diagnostic and preparative downstream processes. Amongst these methods, plasmid DNA extraction was the Materials and Methods first to be reported [1] using the tedious alkaline extraction protocol. Investigation of miniprep buffers This involved lysozyme treatment to weaken the Escherichia coli (E.coli) cell wall prior to cell lysis and selective denaturation of genomic Proprietary buffers: Proprietary equilibration buffers (P-BK1 and DNA using sodium dodecyl sulfate (SDS) and sodium hydroxide. P-BK2); resuspension buffers (P-P1); lysis buffers (P-P2); neutralization Sodium acetate is then used to neutralize the alkaline pH, resulting buffers (P-P3-1 and P-P3-2); binding buffer (P-W1); wash buffers (P- in the formation of an insoluble network of denatured genomic W2-1 and P-W2-2) and elution buffers (P-EB1, P-EB2, and P-EB3) for DNA, protein-SDS complexes and high molecular weight RNA. These plasmid extraction with the following ingredients were prepared: complexes were then removed by high speed centrifugation, leaving the P-BK1 ----- NaCl, MOPS desired plasmid DNA in the supernatant [1]. P-BK2 ----- NaOH (> 1M [Na+] than P-BK1) As the protocol was labour-intensive, efforts to simplify the extraction methods gave rise to the development of the "Guanidinium P-P1 ----- Tris Base, EDTA, RNase A Thiocynate - Phenol - Chloroform" method [2] to separate the various P-P2 ----- SDS, NaOH biomolecules through multiple liquid phases [3]. Further developments resulted in doing away with the use of hazardous chemicals (phenol and P-P3-1 ----- C2H3KO2 chloroform) through the use of spin columns for rapid extraction of P-P3-2 ----- NH2C(=NH)NH2 • HCl , C2H3KO2 ( pH< P-P3-1) high purity nucleic acids. Despite simplifying the process through the immobilization of plasmid DNA to the solid phase matrix (i.e. silica), plasmid extraction is still underlined by the need to disrupt bacterial cell walls, denaturation of nucleic acid binding proteins, inactivation *Corresponding author: Samuel Ken-En Gan, Bioinformatics Institute, Agency for of nucleases such as RNases, washing away of undesired contaminants, Science, Technology, and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, 138671, Singapore, Tel: 65-6478-8417, 65-6407-0584; Fax: 65-6478-9047; E-mail: and elution of desired plasmid DNA. [email protected] At the crux, the silica solid phase matrix determines the resultant Received April 25, 2014; Accepted June 06, 2014; Published June 12, 2014 product purity and yield. For optimal DNA binding, equilibration of Citation: Poh JJ, Gan SKE (2014) The Determination of Factors Involved in these silica columns by Na+ is required to break hydrogen bonds for Column-Based Nucleic Acid Extraction and Purification. J Bioprocess Biotech 4: the formation of salt bridges, allowing for spatial interaction with the 157 doi: 10.4172/2155-9821.1000157 negatively charged DNA. The silica membrane is then washed with Copyright: © 2014 Poh JJ et al. This is an open-access article distributed under ethanol to remove salts and other contaminants prior to elution of the the terms of the Creative Commons Attribution License, which permits unrestricted bound DNA using low ionic strength (pH ≥ 7) buffers [3]. use, distribution, and reproduction in any medium, provided the original author and source are credited. J Bioproces Biotechniq ISSN:2155-9821 JBPBT, an open access journal Volume 4 • Issue 3 • 1000157 Citation: Poh JJ, Gan SKE (2014) The Determination of Factors Involved in Column-Based Nucleic Acid Extraction and Purification. J Bioprocess Biotech 4: 157 doi: 10.4172/2155-9821.1000157 Page 2 of 5 P-W1 ----- NH2C(=NH)NH2 • HCl, Isopropanol A and B gel extraction kits and proprietary buffers: Comparisons between gel extractions buffers from generic A, generic B, and the P-W2-1 ----- Tris Base, Ethanol proprietary buffers (P-QG2, P-QG3) were performed in triplicates. P-W2-2 ----- Ethanol Gel protocols for generic A and B (see Supplementary Material) were carried out according to respective manufacturer’s instructions with P-EB1 ----- Tris-Base the exception of standardizing gel dissolution temperature to 60°C P-EB2 ----- Tris-HCl, EDTA and elution of DNA at 35 µl. Gel extractions using proprietary buffers were carried out according to generic A protocol with the exception P-EB3 ----- NaCl, Tris-Base, Isopropanol of varying the ratio of buffer to gel slice to 3:1 w/v ratio (according to Generic brand A (an "original equipment manufacturer" or "OEM" generic B protocol). Time taken for the gel slices to dissolve completely brand) and generic brand B (well-established brand) buffers were were measured with a lab timer and analyzed statistically. purchased from the commercial vendors. Optimization of PCR purification buffers Culturing of Escherichia coli for miniprep: Luria–Bertani (LB, Polymerase chain reaction: PCR reactions of 325 µl were Biopolis Shared Facilities, BSF, A*STAR) with ampicillin (GoldBio, performed containing 6.5µl of Taq polymerase, 19.5 µl 30 mM MgCl2 USA) was used as growth medium. Previously made competent E. and 32.5 µl of 10X PCR Buffer (Axil Scientific), 26 µl of 2 mM dNTPs coli [4] were transformed with ampicillin resistant plasmids bearing mix (Quintech Life Sciences), 13 µl of reverse primer : OriPNrul R antibody genes as previously described [5], and inoculated in LB (5’-ATA TCT CGC GAA TGC TGG GGG ACA TGT ACC TC-3’), ampicillin broth in overnight cultures at 37°C in a shaking incubator. forward primer OriPNrul F (5’-CAC ACT CGC GAA GGA AAA The plasmids were used for miniprep, gel extractions, and PCR GGA CAA GCA GCG AA-3’), template plasmid DNA, and 201.5 µl of amplification. For comparisons, the same plasmids and bacterial HyClone water (Thermo Scientific, Cat no. SH30538.01). The amplicon cultures were used. oriP is ~1.9 kb. The completed PCR mix was transferred into PCR tubes Establishing the OPT and HM buffers with generic brand A of 25 µl aliquots and carried out in Arktik Thermal Cycler (Thermo miniprep kit: To establish the best "home-made" (HM) proprietary Scientific) with the following profile: Initial denaturation at 94°C for buffers, we evaluated the solution by systematically displacing the 5 minutes; 30 cycles of denaturation at 94°C for 1 minute, annealing buffers in kit A while following its protocol (Supplementary Material). and extension at 71°C for 3 minutes; and final extension at 72°C for For the selection of optimized (OPT) buffers, we chose the best buffers 10 minutes. between our HM buffers and generic A. Comparisons were performed Comparisons of PCR purifications of generic A and B, and in triplicate minipreps, standardizing the use of 4 mL overnight E. coli proprietary buffers: Generic A and B PCR purifications and selected culture, and 40 µl of buffer for elution. The HM and OPT buffers were proprietary buffers from miniprep (P-W1) and gel extraction (P-QG2) selected based on DNA concentration, and A260/280 ratio (Supplementary buffers were carried out in triplicates using the respective generic spin Data). columns. A and B PCR purifications were performed according to Comparison of HM and OPT buffers on generic A and B the respective manufacturer’s recommendations (see Supplementary miniprep kit: Plasmid extraction using HM buffer, OPT buffers, generic Material). The PCR purification using proprietary buffers were carried A and B plasmid extraction kit were each carried out in triplicates. out using generic A’s protocol, with the exception of using 5:1 volume All commercial kits were used according to the manufacturer's ratio of buffer to PCR reaction (according to generic brand B protocol). recommendations (see Supplementary Material). Comparison of DNA analysis HM and OPT buffers were performed according to the commercial protocol with the exception of varying the buffers on spin columns of DNA concentration and A260/280 ratio were analysed both generic A and B. spectrophotometrically using IMPLEN Nanophotometer P330 in triplicates. 1% TAE agarose gels were used to analyse quantity and quality Investigation of gel extraction buffers of plasmid DNA extracted from the minipreps and PCR purifications. Preparation of proprietary buffer for gel extraction: Proprietary 10 µl of extracted/purified DNA from the above comparisons were gel dissolving buffers (P-QG2, P-QG3) for gel extraction were loaded with 6x loading dye containing SYBR Green (Quintech Life prepared with the following ingredients: Sciences) and analyzed using the RunVIEW electrophoresis apparatus (Cleaver Scientific). P-QG2 ----- NH2C(=NH)NH2 • HSCN, Tris-HCl, EDTA Statistical analysis P-QG3 ----- NH2C(=NH)NH2 • HSCN, C6H13NO4S • xH2O Time taken for the gel dissolution, DNA concentration and A260/280 Only the gel dissolution buffers were investigated as the other ratio from the nucleic acid extractions were analyzed using One-Way buffers were previously determined in the miniprep comparisons. ANOVA and independent T-tests. Significance were deemed when Electrophoresis and excision of gel fragment: 1% TAE agarose gel p<0.05. All statistical analysis was performed using SPSS 17.0 (IBM). was used for running the same volume of plasmids in triplicates with 6x loading dye containing SYBR Green (Quintech Life Sciences Pte Ltd, Results and Discussion Singapore). Gel bands were excised with a fixed volume gel cutter and From the systematic testing, we established a set of optimized weighed using Mettler Toledo analytical balance. The gel slices typically (OPT) and completely "home-made" (HM) buffers for nucleic acid weighed between 200-300 mg. extraction and purification kits that are comparable to the two generic brands (A and B) in terms of plasmid yield and purity. Through the Comparison of dissolving rate and DNA recovery of generic step-wise buffer substitution (Table 1), we found that P-P1, P-W2-1 J Bioproces Biotechniq ISSN:2155-9821 JBPBT, an open access journal Volume 4 • Issue 3 • 1000157 Citation: Poh JJ, Gan SKE (2014) The Determination of Factors Involved in Column-Based Nucleic Acid Extraction and Purification. J Bioprocess Biotech 4: 157 doi: 10.4172/2155-9821.1000157 Page 3 of 5 Buffer OPT buffer HM buffer benchmarking was performed against the generic brand A and B Buffer type No of expts comparisons selection selection miniprep kits. Generic brand B was specifically picked due to the No buffer < GA-BK 3 of 3 company’s reputation in these kits. Our comparisons (Figure 1) found Equilibration P-BK1 < GA-BK 4 of 6 GA-BK P-BK2 that the use of HM buffers on generic A columns did not perform P-BK2 = GA-BK 2 of 2 better than generic A buffers. This was expected since some HM buffers Resuspension P-P1 > GA-A1 3 of 4 P-P1 P-P1 (equilibration, lysis and neutralization) gave poorer yields than generic Lysis P-P2 < GA-A2 3 of 4 GA-A2 P-P2 A counterparts (Table 1). P-P3-1 < GA-A3 3 of 4 Neutralization GA-A3 P-P3-2 When OPT buffers were used on generic A spin columns (top panel P-P3-2 < GA-A3 2 of 4 Pre-wash P-W1 = GA-W1 2 of 4 P-W1 P-W1 of Figure 1, lane 2), and HM buffers on generic B spin columns (top P-W2-1 > GA-W2 3 of 4 panel of Figure 1, lane 6), we found that OPT buffers had higher yields Wash P-W2-1 P-W2-1 than both generic A and B kits when using their respective buffers (top P-W2-2 < GA-W2 2 of 4 P-EB1 = GA-EB 2 of 4 panel of Figure 1, lanes 1 and 4, respectively). HM buffers on generic Elution P-EB2 > GA-EB 3 of 4 P-EB2 P-EB2 B spin columns had the highest yields despite yielding the least DNA P-EB3 < GA-EB 3 of 3 on generic A spin column. This shows that generic B spin columns had superior DNA binding capability, and that OPT buffers would give [DNA] were measured in three separate readings using IMPLEN Nanophotometer P330. Independent T-test was used to determine the statistical significance of the the best yields since they outperformed generic A buffers, which were differences between test buffers and generic brand A buffers. Differences in [DNA] in turn, superior to HM buffers (top panel of Figure 1). As the exact were determined when p<0.05. differences between generic A and B spin columns were unknown to Table 1: Comparison between the proprietary buffers and generic brand A miniprep us, we are unable to discuss this further. kit buffers using DNA yields. On the fastest gel dissolution time, P-QG2 buffer showed the fastest and P-EB2 were buffers capable of obtaining high DNA yields. average rate (211 secs), followed by P-QG3 (220 secs), generic B QG On the equilibration of the spin columns (using generic brand A), (234 secs), and generic A G-G1 buffer (354 secs; see Figure 2). ANOVA the use of GA-BK buffer expectedly gave higher DNA yields than "no tests showed that the time differences were significant (F (3, 32) = buffer" conditions (Table 1). Since the equilibration buffers contained 129.86, p=0.000). Na+, a salt bridge could be formed, permitting DNA adsorption onto On DNA recovery, P-QG2 had better or similar DNA recovery the silica particles [3]. We found that our P-BK buffer yielded less compared to both generic A and B buffers, respectively (Table 2), DNA compared to GA-BK (generic A) and P-BK2 buffer, as the latter whereas P-QG3 buffer had lower yields than generic brand B kit two buffers had higher concentrations of Na+ (more than double despite being comparable to brand A. Investigations between our two the molarity), this demonstrates that high [Na+] allowed for effective proprietary buffers showed that P-QG2 had higher concentrations equilibration. of guandidine thiocyanate (by almost 1 M). As a chaotropic agent Regarding the neutralization buffers, P-P3-2 buffer yielded that removes DNA binding proteins [7], the higher concentration significantly higher DNA than the P-P3-1 buffer (t (70) = 2.121, p=0.038, see Table 1). Since both P-P3-1 and P-P3-2 buffers acted to neutralize the alkaline lysis buffer, the resultant pH after neutralization GEN A OPT A HM-A GEN B OPT-B HM-B would be lower for P-P3-2 due to its stronger acid component, 10 kb supporting previous reports that lower pH (<7) facilitated better DNA- 8 kb silica adsorption [3]. Amongst the wash buffers, P-W2-1 buffer had the highest DNA GEN A HM-A OPT-A GEN B HM-B OPT-B yield while P-W2-2 buffer had the lowest yields. From studying buffer recipes, we propose that the higher salt content in P-W2-1 increased the Nicked stringency of the column washes, removing nucleases more efficiently Supercoiled and preventing DNA degradation. GEN A HM-A GEN B HM-B On the comparison of the elution buffers, P-EB2 was significantly better than P-EB1 (t (64) = 2.19, p = 0.032). As the only elution buffer PCR with EDTA, Mg2+, a co-factor in many nucleases [6] would have been Amplicon chelated. Since P-EB-2 also had a hundred-fold more Tris than P-EB1, Figure 1: Final comparison of generic brands A, B, HM and/or OPT miniprep there would be better pH buffering without producing free radicals (top), gel extraction (middle) and PCR Purification (bottom) protocols. that would otherwise speed up the auto-catalytic activity of DNA [6]. GEN A = Nucleic acid extractions using generic A kits; Comparatively, P-EB3 showed the lowest DNA yields (even below that HM-A = Nucleic acid extractions using generic A spin column with HM buffers; OPT-A = Nucleic acid extractions using generic A spin column with OPT buffers; of generic A buffer). Since isopropanol was a component, it is likely GEN B = Nucleic acid extractions using generic B kits; that DNA precipitation may have occurred, lowering yields. HM-B = Nucleic acid extractions using generic B spin column with HM buffers; OPT-A = Nucleic acid extractions using generic B spin column with OPT buffers. As generic A and B buffer recipes were not known to us, we were 10uL of DNA extracted were mixed with 6x loading dye and loaded on a unable to discuss the likely factors that contributed to the different 1% TAE agarose gel. All DNA concentrations determined using IMPLEN Nanophotometer P330 were signifcantly different between groups in One-Way DNA yields observed for the resuspension, lysis and pre-wash buffers ANOVA. F (5, 12) = 109, p = 0.000, for miniprep; F (5, 12) = 170.691, p = 0.000, (P1, P2, and W1, respectively). for gel extraction; and F (3, 8) = 1250.141, p = 0.000, for PCR Purification. Please see supplementary data for nanospectrophotometer readings. With the HM and OPT buffer components determined, J Bioproces Biotechniq ISSN:2155-9821 JBPBT, an open access journal Volume 4 • Issue 3 • 1000157 Citation: Poh JJ, Gan SKE (2014) The Determination of Factors Involved in Column-Based Nucleic Acid Extraction and Purification. J Bioprocess Biotech 4: 157 doi: 10.4172/2155-9821.1000157 Page 4 of 5 of guanidine would have aided in better adsorption to the silica gels. (A) Being similar to other chaotropic agents (e.g. potassium or sodium iodide), which are necessary for dissolving agarose gels [8], the higher 35 concentrations of guanidine thiocyanate would also dissolve the 30 Average DNA Concentration (ng/µL) agarose quicker. Thus, on the basis of timing and yields, P-QG-2 was 25 chosen as the optimal buffer. 20 For PCR kits, we compared only the PCR binding buffers i.e. the 15 optimized P-W1 and P-QG2 with generic A and B buffers. ANOVA tests showed significant differences between the DNA recovered, F 10 (5, 48)=261.72, p=0.000. It was observed that using generic B column, 5 buffer P-W1 (P-W1-B in Figure 3A) obtained the highest DNA 0 recovery, almost up to 30 ng/µL. On the contrary, the same P-W1 Gen A P-QG2-A P-W1-A Gen B P-QG2-B P-W1-B buffer in generic A column yielded only slightly above 20 ng/µL, Buffers thus supporting previous miniprep observations that generic B spin Replicate 1 Replicate 2 Replicate 3 columns were superior with respect to DNA binding. Normalizing the spin columns by comparing P-W1 on both A and B spin columns, Figure 3A: Comparison of the DNA recovered using P-QG2, P-W1, generic brands A and B’s PCR purification buffers. buffers of both generic brands would generate similar yields. Bar chart showing the means and standard errors of the DNA recovered using Electrophoresis of the purified PCR products using the different various buffers. P-QG2-A and P-W1-A buffer testing were carried out using brand A’s protocol (see Supplementary Material). On the other hand, P-QG2-B and P-W1-B buffers were performed on brand B’s spin column (without P-BK). Buffer comparisons Total no of expts DNA concentrations were determined by IMPLEN Nanophotometer P330 in three separate extractions measured in triplicates. P-QG2 > Generic A 3 of 3 Gen A = PCR Purification using generic A PCR Purification Kit; P-QG3 = Generic A 3 of 3 P-QG2-A = PCR Purification using generic A column with P-QG2 buffer; P-QG2 = Generic B 2 of 3 P-W1-A = PCR Purification using generic A column with P-W1 buffer; P-QG3 < Generic B 3 of 3 Gen B = PCR Purification using generic B PCR Purification Kit; P-QG2-B = PCR Purification using generic B column with QG2 buffer; Gel Extractions using P-QG2 and P-QG3 were carried out using generic brand A P-W1-B = PCR Purification using generic B column with P-W1 buffer. protocol (see Supplementary Material) with the exception of varying the gel dis- *p < 0.05; **p < 0.001 for the ANOVA tests comparing the DNA recovered. solving buffer GA-G1. DNA concentrations were measured in three separate readings using IMPLEN Nanophotometer P330. Independent T-tests were used to determine the statisti- spin columns revealed two distinct bands that corresponded to the cal significance of the differences in DNA recovered. Differences were deemed significant when p<0.05 OriP product and primer dimers. Interestingly, P-W1 removed primer Table 2: Comparison of DNA yields in 3 independent gel extraction experiments dimers when used on generic A columns but not on generic B columns. using P-QG2 and P-QG3 buffers compared against generic brands A and B buffers This was likely due to the higher binding capability of generic B using their respective kits and protocols. columns. Nonetheless, P-QG2 was clearly the better buffer as it yielded the highest intensity band regardless of the spin column used (Figure 3B and 3C). 450 Analysis of the P-QG2 and P-W1 recipes found that higher 400 concentrations of guanidine (by ~1 M in P-QG2) resulted in better Time Taken for Gel to Dissolve (Seconds) 350 purification, which we propose to result from the release of polymerases 300 from DNA, allowing their adsorption to the silica. 250 As a final comparison, we carried out trials comparing the full set of HM buffers and OPT buffers against both generic A and B (Figure 200 1). As can be observed, OPT buffers had the best yields regardless of the 150 columns used, with HM buffers comparable to the commercial brands 100 A and B. 50 Conclusion 0 The findings of the study allowed us to rely on more cost-effective Experiment 1 Experiment 2 Experiment 3 Gen A Buffer Gen B Buffer P-QG2 Buffer P-QG3 Buffer columns without compromising experiments. Extending beyond the kits tested, the factors of these buffers also underline processes such as Figure 2: Comparison of time taken for excised gel fragments to dissolve midi, maxi and giga scale DNA extractions, allowing labs to optimize completely using generic brand A and B gel dissolving buffers, P-QG2 and P-QG3 buffer. their own cost-effective reagents by the addition of important chemicals Bar chart showing the means and standard errors of the time taken for excised to their existing buffers or kits (e.g. adding a Na+ column equilibration gel fragments to dissolve completely using gel dissolution buffers of generic A and B, P-QG2 and P-QG3 in 3 independent experiments. Gel extractions step to existing commercial kits). Through detailed analysis of buffer using P-QG2 and P-QG3 were carried out using brand A’s protocol (see constituents, we were able to validate the importance of: Supplementary Material) with the exception of varying the gel dissolution buffer GA-G1. Time taken for each excised gel to dissolve completely were 1) Na+ concentrations in column equilibration. determined with a timer. Gel fragments weighed around 200-300 mg and respective buffers used to dissolve gel were aliquoted accordingly. *p < 0.05; 2) Importance of strong acids for low pH in the neutralization of **p < 0.001 for the One-Way ANOVA. cell lysis buffer. J Bioproces Biotechniq ISSN:2155-9821 JBPBT, an open access journal Volume 4 • Issue 3 • 1000157 Citation: Poh JJ, Gan SKE (2014) The Determination of Factors Involved in Column-Based Nucleic Acid Extraction and Purification. J Bioprocess Biotech 4: 157 doi: 10.4172/2155-9821.1000157 Page 5 of 5 (B) GEN A P-QG2-A P-W1-A PCR Amplicon 250 bp (C) GEN B P-QG2-B P-W1-B PCR Amplicon 250 bp Figure 3B and 3C: Comparison of the P-QG2 and P-W1 with generic brand A and B PCR purification kits. (B) Comparisons of P-QG2 and P-W1 buffers were performed using generic A spin columns. (C) Comparisons of P-QG2 and P-W1 buffers were performed using generic B spin columns. 10uL of purified PCR DNA were mixed with 6x loading dye and loaded on a 1% TAE agarose gel using RunVIEW by Cleaver Scientific. Samples were processed from the same PCR reaction for consistency. 3) High salt for higher stringency in column washes. 1. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7: 1513-1523. 4) Tris and chelating agents to remove nuclease cofactors and pH 2. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid buffers that would not generate free radicals. guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159. 5) High presence of chaotropic agents for faster gel dissolution and 3. Tan SC, Yiap BC (2009) DNA, RNA, and Protein Extraction: the Past and the removal of interfering proteins for both gel and PCR kits. Present. Journal of Biomedicine and Biotechnology 2009: 1-10. Acknowledgement 4. Chan WT, Verma CS, Lane DP, Gan SK (2013) A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Biosci We would like to thank Sir David Lane, Chandra Verma, Peck-Ting for their Rep 33. administrative support, without which the work would not be possible. We also thank Mr Keane MJ Lim for assisting in the formatting of the article. This work 5. Gan S, Hunt J, Marsh P, Beavil A, Harries H (2009) The design and is mainly funded by Quintech Life Sciences Pte Ltd, with support from the Joint optimisation of a transient expression system for the rapid expression of human Council Office, Agency for Science, Technology, and Research, Singapore. immunoglobulin E. Competing Interests 6. Gerstein AS (2004) Molecular Biology Problem Solver: A Laboratory Guide. John Wiley & Sons, New York, USA. This work was commissioned by Quintech Life Sciences Pte Ltd to explore the factors to making better buffers, which may be made commercially available. There 7. Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-Van Dillen P, et are no other competing interests. al. (1989) Rapid and Simple Method for Purification of Nucleic Acids. J Clin Microbiol 28: 495-503. References 8. McPherson M, Moller S (2006) Purification and Cloning of PCR Products, PCR: Taylor and Francis Group, New York, USA. 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