SMOBiO Product information GetClone PCR Cloning vector CV1000 GetClone PCR cloning vector, 20 rnx CV1001 GetClone PCR cloning vector, 40 rnx Certification of analysis The GetClone was tested for cloning a 959 bp amplicon with T4 DNA ligase. Aliquot of 5 μl ligation mixture was used to transform 50 μl of chemically competent JM109 with puc19 transform efficiency was 5x10 7 cfu/μg. Cloning with efficiency exceeded 0.6 10 5 cfu/μg. Greater than 90% cloning efficiency was achieved by GetClone cloning vector incorporating a 956bp control amplicon. Contents Page Component of the GetClone vector... 1 Storage... 1 Description.... 1 Cloning principle.. 1 Important note.. 2 Cloning protocols..... 3 Analysis of recombinant clones.. 4 Map and features of GetClone vector... 5 Genetic elements and restriction enzyme map... 6 Troubleshooting.... 7
Component of the GetClone vector The package 20 Rxn 40 Rxn pget1.1 vector 23 µl 46 µl pget-for primer, 10 µm 100 µl 200 µl pget-rev primer, 10 µm 100 µl 200 µl Primers sequence: pget-for: 22-mer 5'-TCGAAGTTAAAGATGATTACGG-3' pget-rev: 22-mer 5'-TCTCTCGATAGCATTTCCTGC-3' Storage: All components of the GetClone PCR cloning vector should be stored at -20 Desciption: The GetClone vector is a positive selection system for the high efficiency cloning of blunt end amplicons generate with Pfu, KOD plus or other proofreading DNA polymerase. This cloning vector contains a lethal restriction endonuclease gene which can be disrupted by ligation of a blunt end DNA insert into the cloning site. Only colonies with inserted vectors are able to propagate, eliminating the additional needs of IPTG and X-Gal for blue/white screening. The GetClone vector contains multiple cloning sites BamHI, EcoRI, SpeI, SnaBI, BamHI and PstI for insert release. Sequencing primers are also included for convenient colony PCR or sequencing of the cloned insert. Cloning principle: GetClone vector can accept inserts from 6 bp to 12 kb. Because the 5' end of the vector contains the phosphoryl groups, the phosphorylation of PCR fragment is not required. The blunt amplicon generated by proofreading DNA polymerase can be directly ligated follow personal ligation system. It is not recommended to use the non-proofreading DNA polymerase which may result in sequence errors during PCR reaction, especially when amplifying long DNA fragments. 1
Important notes 1. This package doesn t include the ligation system. The cloning vector is compatible with regular ligation system available on the market. 2. Make sure all the tubes or eppendorf is nuclease free. 3. This GetClone vector only accepts blunt end PCR product or blunt DNA fragment not for the sticky end DNA fragment. 4. Analyze the PCR product by electrophoresis and elute the target fragment before ligation. 5. Mix the insert and the GetClone vector by gently pipetting the aliquot before ligation. 6. UV radiation and EtBr will reduce ligation efficiency 1, 2. For efficient cloning of the gel-purified DNA fragment, to avoid DNA damage caused by low wavelength UV radiation, we recommend using the SMOBiO B-BOX Blue Light LED epi-illuminator (VE0100 ) and ExcelStain DNA Fluorescent Staining Dye (DS1000, DS1001). Otherwise to limit the time of DNA exposure to UV light for few seconds is recommended. 7. GetClone was recommended for using a 3:1 molar ratio of the control insert/vector, but also capable to accept a wide range of insert/vector ratio (0.5:1 to 12:1). 2
Cloning protocols The molar ratio: It is recommend 3:1 of insert to vector Ex: For 1kb fragment at 50 ng/µl The insert/vector ratio= 50 ng/1kb (insert size): 50ng/3kb (vector size) =3:1 The ligation reaction: Use the NEB T4 DNA ligase as example 1. Component Volume PCR product 1 µl (50 ng/µl) GetClone vector 1 µl (50 ng/µl) Ligation buffer 2 µl Water, nuclease free Up to 20 µl T4 DNA Ligase 1 µl 2. Incubate at room temperature (20-25 ) for 10 min to one hour or 16 for overnight. For longer DNA we will recommend to do ligation at RT for one hour. Otherwise ligation condition may be adjusted according to manufacturer s instruction of the used ligation system. 3. Transform chemically competent E. coli cells with 10 µl of the ligation mixture or electrocompetent cells with 1 µl of de-salt ligation mixture. 4. At least 90% analyzed colonies should contain recombinant plasmid when using the 959 bp insert. 5. It is recommended using competent cell with efficiency greater than 1x10 6 cfu/µg DNA. 3
Analysis of recombinant clones 1. By restriction enzyme digestion Isolate the plasmid DNA from bacterial culture of the target colonies. The BamHI was recommended for release the insert DNA from the vector. 2. By colony PCR i. Pick target colonies (0.5-1 mm diameter) individually with sterilized toothpick and rub on the bottom of PCR tube. ii. Prepare the PCR master mix for the total number of colonies analyzed plus one extra to compensate pipetting error. For each 50 µl reaction, mix the following reagents: Component Using ExcelTaq DNA polymerase (#TP1000) Using ExcelTaq 5X PCR Master Dye Mix (#TP1302) 10X Taq buffer 2.5 µl dntp mix, 2.5mM each 2.0 µl pget-for primer, 10 µm 0.4 µl 0.5 µl pget-for primer, 10 µm 0.4 µl 0.5 µl Nuclease free water 19.5 µl 19.0 µl Taq DNA polymerase 0.2 µl 5X PCR master mix 5 µl Total volume 25 µl 25 µl iii. Mix well. Transfer 25 µl mixture into the PCR tubes. iv. Perform PCR program: 94, 4 min; 94,30 s, 55,20 s,72 30 s/kb for 32 cycles; 72 1 min. v. Analyze the PCR product on an agarose gel. 3. By sequencing Both provided pget-for primer or pget-rev primer are suitable for sequencing. 4
Map and features of GetClone vector The GetClone vector includes linearized pget1.1 plasmid illustrated in Figure 1. The pget1.1 vector has been linearized by restriction enzyme digestion (Fig. 2) to present two blunt ends; thus retaining the 5'-phosporyl group at both 5' end. The nucleotide sequence is available at www.smobio.com. Fig. 1. pget1.1 map 5
Fig. 2. DNA sequence of MCS region Genetic elements of the pget1.1 vector Element Function Position (bp) Leghal gene For screening against self-ligation 1...834 MCS Multiple cloning site 278...412 Insertion site The ligation site of blunt end insert 354...355 M13 Rev M13 reverse primer 838...854 puc-ori Initiation of replication 1022...1610 P The promoter for expressing the 2011...2115 ampicillin resistance and lethal gene Amp R Ampicillin resistance gene 2115...2973 M13 for M13 forward primer 2975 2990 The restriction enzyme that cut GetClone once or twice Enzyme The cut positions Enzyme The cut positions AclI 2302, 2675 FspI 2681 ApaLI 1353, 2232 HindIII 204 AvaII 2540, 2762 HpaI 568, 822 BamHI 279, 375 NspI 4, 1671 BanI 1923, 2951 PciI 1667, 2995 BglII 434 PssI 1935 Bme1580I 1357, 2236 PstI 418 BsaAI 368, 791 PvuI 2535 BsaHI 2364 PvuII 1847 BseYI 1363 ScaI 2423 BspHI 947, 2064 SnaBI 368 BsrDI 2670, 2844(c) SpeI 322 BsrFI 2819 SspI 2099 CdiI 2351, 2652 (c) StyI 718 EcoRI 315 TatI 2421 6
The restriction enzymes that do not cut GetClone vector No cuts: AatII, AccI, AfeI, AflII, AgeI, ApaI, AscI, AsiSI, AvaI, AvrII, BanII, BbvCI, BclI, BlpI, BmgBI, BmtI, Bpu10I, BsaBI, BsiWI, BsmI, BspEI, BspMI, BsrGI, BssHII, BstBI, BstEII, BstZ17I, Bsu36I, BtgI, ClaI, EagI, EcoNI, FseI, FspAI, KpnI, MfeI, MluI, MscI, NaeI, NarI, NcoI, NdeI, NgoMIV, NheI, NotI, NruI, NsiI, PacI, PfoI, PmeI, PmlI, PpuMI, PsiI, RsrII, SacI, SacII, SalI, SanDI, SbfI, SexAI, SgrAI, SmaI Troubleshooting Problem Background colonies without correct inserts Background colonies without insert Sequence errors in the cloned insert Few or no transformants Solution and cause The PCR products contain non-specific amplicon or primer dimmers were cloned. If the electrophoresis indicates multiple fragments, it is suggested to perform the gel elution before the ligation. False-negative in colony PCR It is recommended that the diameter of colony should around 0.5-1 mm when doing the colony PCR. Colonies that are too small or too big may affect colony PCR. DNase contamination Nuclease contamination may cause the frameshift of lethal gene, thus disabling the positive selection. 1. Low fidelity DNA polymerase was used It is recommended to use high fidelity DNA polymerase to reduce PCR error. 2. Errors in PCR primers If the sequence errors were found in primer region, this may be cause by the primer degradation or error during primer synthesis. We recommend re-ordering primers from a reliable supplier. 1. Inadequate transformation efficiency of competent E. coli cells 2. Sticky-end PCR product was used in the ligation process without blunting. 3. Insert/vector ratio is outside of the optimal range. 4. PCR products was damaged by UV light Cloned sequence is lethal to E coli. 7
Reference 1. Boyce, R.P. and Howard-Flanders, P. (1964) Release of ultraviolet-light induced thymine dimers from DNA in E. coli K-12. Proc. Natl. Acad. Sri.USA, 51, 293-300. 2. Cariello, N. F., Keohavong, P., Sanderson, B. J. S. and Thilly, William G. (1988). DNA damage produced by ethidium bromide staining and exposure to ultraviolet light. Nucleic Acids Research,16,4157. Product use limitation This product is designed for research purposes only and not certified for use in diagnostic or any clinical application. 8