pGEM-T Vector Systems Troubleshooting guide

Promega Notes Magazine Number 45, 1994, p.19 pGEM®-T Vector Systems Troubleshooting Guide Jacqui Robles and Mary Doers Promega Corporation The pGEM-T Vector System allows direct cloning of PCR* products without restriction digestion, modification of primers, or purification of amplified DNA. Introduction Of the several methods currently employed for cloning PCR* products, most require extra manipulation of the DNA after amplification. One method involves using oligonucleotide primers that contain restriction sites within their sequence. This approach not only increases the cost of primer synthesis but is also inefficient - cutting with restriction enzymes near the end of PCR products has proven to be very difficult (1). Another approach is to enzymatically treat the PCR product to produce blunt ends and then ligate the fragment into a blunt-ended vector. PCR products do not have blunt ends because Taq DNA Polymerase** catalyzes the addition of nucleotides, almost exclusively adenosines, in a non-template directed manner to the 3´-termini of the double stranded products (2). In addition to requiring extra steps, blunt-end ligations are inefficient and so this approach requires a high concentration of blunt-ended PCR product. *The polymerase chain reaction (PCR) process for amplifying nucleic acid is covered by U.S. Pat. Nos. 4,683,195 and 4,683,202, assigned to Hoffmann-LaRoche. Patents pending in other countries. ** This product has not been licensed for use in the polymerase chain reaction (PCR). The pGEM®-T Vector Systems take advantage of the non-template dependent addition of a single deoxyadenosine to the 3´-end of PCR products by using a vector with ends that complement those of the amplified fragment. The pGEM-T Vector is prepared by digesting Promega's pGEM-5Zf(+) Vector with EcoR V to generate blunt ends and then adding a terminal thymidine to the 3´-strand at both ends. These single 3´-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid by providing ends complementary to those of the insert and reducing self-ligation. The system requires very little starting material, and in most cases, fragments can be cloned directly without further manipulations. Controls assure product performance To assure good performance of the system, use only the components provided with the system. The T4 DNA Ligase supplied has been quality tested by ligating the Control DNA, a 469 base pair PCR product, into the pGEM-T Vector. Low quality or nuclease contaminated ligase may promote the loss of the overhanging T's in the pGEM-T Vector, which in turn favors the blunt-end self-ligation of the vector over the ligation of the vector with the insert. The competent cells supplied with the system should be handled as recommended to preserve their high efficiency: 10^8 cfu/µg of DNA. When used as recommended, the percentage of white colonies with the system's Control DNA insert should be at least 15%, of which 80% are usually true recombinants. The percentage of recombinant colonies obtained when cloning other inserts (in a size range of 500 to 1000 base pairs) may be as high as 80%, depending on the composition and purity of the fragment. Several important factors that should be considered when cloning in the pGEM-T Vector are described in Table 1.

Table 1. Troubleshooting Guide for the Use of pGEM-T Vector.

ResultsPossibleCausesRecommendations

--------------------------------------------------------------------------LigatingtheControlDNAInsert

--------------------------------------------------------------------FewornoCellshavelostTesttheefficiencyofthecolonies.competency.cellsbytransformingan

uncutplasmidthatencodesantibioticselection,forexamplepGEM-5Zf(+).

--------------------------------------------------------------------HighbackgroundVectorself-ligatedUseonlytheT4DNALigaseofbluecolonies.withoutT's.providedwiththekit.It

hasbeenqualitytestedforuseinthepGEM-TVectorsystem.

--------------------------------------------------------------------------LigatingaPCRinsert

--------------------------------------------------------------------LowpercentageofVectorself-ligatedUseonlytheT4DNALigasewhitecolonies.withoutT's.providedwiththekit.

--------------------------------------------------------------------InsufficientamountAdjustthevector:insertofinsertavailable.ratio.

-------------------------------------------------PCRproductlessthanTheseinsertsdonot500basepairslongefficientlydisrupttheoramultipleof3lacZgene.Screenblueorbasepairslong.palebluecoloniesforthe

presenceofinserts.

-------------------------------------------------MultiplePCRGelpurifythefragmentofproducts.interestifmultiplebands

weregeneratedduring

amplificationorifthereisalargeamountofdimerpresent.

--------------------------------------------------------------------UnexpectedSecondaryproductsorGelpurifythePCRproductinsertsinwhiteprimersareclonedin(seeabove).colonies.thepGEM-Tvector.

--------------------------------------------------------------------InsertinblueorClonedinashortPCRPCRproductsislessthanpaleblueinsertwhich500basepairslongoracolonies.maintainedlacZinmultipleof3basepairs

readingframe.long.

--------------------------------------------------------------------------

Method to quickly screen pGEM®-T vector recombinants

To determine the percentage of true recombinants, rapidly perform a screen by picking a number of white colonies and resuspending each one in 50µl of sterile water. Boil for 5-10 minutes and spin in a microcentrifuge for 2-3 minutes to pellet cell debris. Use 5-10µl of this supernatant as the template for small PCR reactions. Amplify with the same primers used to generate the insert or primers within the vector such as the T7 Promoter and SP6 Promoter Primers (Cat.# Q5021 and Q5011). Alternatively, screen potential recombinants by growing 1ml liquid cultures for 6 hours to overnight. Extract the DNA using the Wizard(TM) Minipreps DNA Purification System and digest each sample with a restriction enzyme that is characteristic of the insert (ideally, one with a unique restriction site in the insert and no more than one site in the vector). Under certain circumstances, pale blue colonies may contain inserts as well; small inserts and certain reading frames are less efficient in disrupting the lacZ gene, thus the blue/white color differentiation is less pronounced (3). Summary The pGEM-T Vector Systems provide a convenient method for the direct cloning of PCR fragments. The T4 DNA Ligase included with the systems has been quality controlled to provide the maximum percentage of recombinant clones. The blue/white cloning assay allows quick screening and identification of recombinant clones. References 1.Kaufman, D. L. and Evans, G. A. (1990) BioTechniques 9, 304. 2.Clark, J. M. (1988) Nucl. Acids Res. 16, 9677. 3.Murray, E., Singer, K., Cash K., and Williams R. (1993) Promega Notes 41, 1. Ordering Information ProductCat.#--------------------------------------------------------------------------pGEM®-TVectorSystemIA3600--------------------------------------------------------------------------ThesystemincludesthepGEM®-TVector,Ligase,10XBufferandaPositiveControl.ProductCat.#--------------------------------------------------------------------------pGEM®-TVectorSystemIIA3610--------------------------------------------------------------------------ThesystemincludesthesamecomponentsasSystemIplushighefficiencyJM109CompetentCells.©1994 Promega Corporation. All Rights Reserved. pGEM is a registered trademark of Promega Corporation. Wizard is a trademark of Promega Corporation.