Protocols and Applications Guide (US Letter Size) - Promega

||||||||||||| 13Cloning
X-Gal (Cat.# V3941) and IPTG (Cat.# V3955). Both the
pGEM®-T and pGEM®-T Easy Vectors contain numerous
restriction sites within the multiple cloning region. The
pGEM®-T Easy Vector multiple cloning region is flanked
by recognition sites for the restriction enzymes EcoRI, BstZI
and NotI, thus providing three single-enzyme digestions
for release of the insert. The pGEM®-T Vector cloning
region is flanked by recognition sites for the enzyme BstZI.
Alternatively, a double-digestion may be used to release
the insert from either vector.
The pGEM®-T and pGEM®-T Easy Vectors also contain the
origin of replication of the filamentous phage f1 for the
preparation of single-stranded DNA (ssDNA). Both
pGEM®-T vector systems include a 2X Rapid Ligation
Buffer for ligation of PCR products, which requires only a
1-hour incubation at room temperature. The incubation
period may be extended to increase the number of colonies
after transformation. Generally, an overnight incubation
at 4°C will produce the maximum number of transformants.
Inserts of several kilobases have been successfully cloned
into the pGEM®-T and pGEM®-T Easy Vectors
(D’Avino et al. 2004). However, as the insert gets larger, the
ratio of vector to insert may need to be optimized further
to maximize ligation efficiency (see Ligation and
Transformation in the section "Vector:Insert Ratio").
One of the disadvantages of PCR cloning into a T vector is
that the insert can be cloned in either direction. Analysis
of recombinant vectors by PCR or restriction enzyme
digestion can be used to determine not only the success of
cloning, but in which direction the insert was cloned. To
verify the direction of the insert, amplify recombinant
plasmids using one of the gene specific PCR primers and
each of the phage promoter primers, which are present on
the pGEM®-T Vector (Knoche and Kephart, 1999). The
correct orientation is important for transcription or
translation or both.
Additional Resources for the pGEM®-T and pGEM®-T
Easy Vector Systems
Technical Bulletins and Manuals
TM042 pGEM®-T and pGEM®-T Easy Vector
Systems Technical Bulletin
(www.promega.com
/tbs/tm042/tm042.html)
Promega Publications
eNotes pGEM®-T Easy Vector System is an easy
tool for preparing gel shift probes
(www.promega.com
/enotes/applications/ap0039_tabs.htm)
eNotes Cloning differential display-PCR products
with pGEM®-T Easy Vector System
(www.promega.com
/enotes/applications/ap0025_tabs.htm)
Protocols & Applications Guide
www.promega.com
rev. 3/09
PN082 Technically Speaking: T-vector cloning
(www.promega.com
/pnotes/82/10203_24/10203_24.html)
PN073 Constructing genomic libraries using the
pGEM®-T Vector
(www.promega.com
/pnotes/73/8235_26/8235_26.html)
PN071 Cloning blunt-end Pfu DNA
Polymerase-generated PCR fragments into
pGEM®-T Vector Systems
(www.promega.com
/pnotes/71/7807_10/7807_10.html)
PN065 Stability of pGEM®-T Vectors
(www.promega.com
/pnotes/65/6921_25/default.html)
PN062 Cloning blunt-end DNA fragments into
the pGEM®-T Vector Systems
(www.promega.com
/pnotes/62/7807_15/promega.html)
PN061 Amplification of flanking regions: New
applications and performance
optimization of single specific primer PCR
and T-vector cloning
(www.promega.com
/pnotes/61/6222_26/promega.html)
Online Tools
pGEM®-T Easy Vector sequence (www.promega.com
/vectors/pgemtez.txt)
pGEM®-T Vector sequence (www.promega.com
/vectors/pgemt.txt)
Citations
Renaud, S. et al. (2007) Dual role of DNA methylation inside
and outside of CTCF-binding regions in the transcriptional
regulation of the telomerase hTERT gene. Nucleic Acids
Res. 35, 1245–56.
Telomeres shorten by 50–100 bases with each cell division,
making the telomere a "mitotic counter" that can limit
cellular lifespan. Telomerase is a two-component protein
consisting of a reverse transcriptase (hTERT) bound to its
own RNA template that can act to maintain telomere length
in dividing cells. This paper investigated the role of
methylation of the hTERT promoter and the transcription
factor CTCF in regulation of telomerase activity. LacZ
reporter plasmids driven by the hTERT minimal promoter
were transiently transfected into HeLa cells, and reporter
assays were performed on lysate generated using Passive
Lysis Buffer. The hTERT minimal promoter did not show
activity if all of the CpG sites were methylated. The
promoter and first exon of hTERT were amplified using
PCR Master Mix from sodium bisulfite-treated genomic
DNA isolated from telomerase-positive cell lines and
tissues. The resulting fragments were cloned using the
pGEM®-T Vector System II. For the methylation cassette
assay, methylated and unmethylated fragments were cloned
into a methylated or unmethylated vector using the
LigaFast Rapid DNA Ligation System. The authors
PROTOCOLS & APPLICATIONS GUIDE 13-4