What is pGEM? The pGEM®-T Vector Systems are convenient systems to clone PCR products generated by certain thermostable polymerases. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the
What is pGEM?
The pGEM®-T Vector Systems are convenient systems to clone PCR products generated by certain thermostable polymerases. These polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments.
What feature makes the pGEM T vector suitable for ligation of PCR products?
The pGEM®-T Easy pre-linearized Vector contains 3´-T overhangs at the insertion site to provide a compatible overhang for PCR products. The provided 2X Rapid Ligation Buffer allows reactions to be completed in 1 hour at room temperature.
What does ligation buffer do?
T4 DNA Ligase catalyzes the formation of phosphodiester bonds in the presence of ATP between double-stranded DNAs with 3´ hydroxyl and 5´ phosphate termini. The unique T4 DNA Ligase buffer optimizes ligation, which can be performed in 5 minutes (1). Single-stranded nucleic acids are not substrates for this enzyme.
What is the principle of TA cloning?
The TA cloning method takes advantage of the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3′-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3′-T overhangs.
What is the pGem-T sequence and map parental vector?
pGEM-T Sequence and Map Parental vector for TA cloning of PCR products. The insertion site is flanked by BstZI sites. This vector is also known as pGEM®-5Zf(+).
What’s the difference between pGem-T and pGem T easy?
The only difference between pGEM-T and pGEM-T Easy is in the multiple cloning site (MCS). The MCS of the pGEM-T Easy Vector contains sequences on either side of the insert that are recognized by the restriction enzymes Not I and EcoR I. This allows the insert DNA to be removed with a single restriction digest using…
Is the pGem-T easy vector available in Addgene?
This vector is NOT available from Addgene. The only difference between pGEM-T and pGEM-T Easy is in the multiple cloning site (MCS). The MCS of the pGEM-T Easy Vector contains sequences on either side of the insert that are recognized by the restriction enzymes Not I and EcoR I.
When to add Sam to the pGem-T sequence?
For full activity, add fresh S-adenosylmethionine (SAM). ApaI can be used between 25°C and 37°C. Sticky ends from different StyI sites may not be compatible. Efficient cleavage requires at least two copies of the SacII recognition sequence.