I attempt to clone the genomic sequence of a couple of isoforms of gene X. When I used the HS PrimeSTAR GXL taq, I could detect very clear bands for four isoforms with the sizes of ~2, ~3, ~6 and ~4.5 kbs (Fig. 1). The fifth isoform shows two distinct bands. My aim is to simply find out the sequence of each isoform and what could be your suggestions in this regard:

1. Our laboratory uses pGEM®-T Easy Vector for routine cloning. But, I wonder that the limitation of insert size bearable by the vector might have a negative impact on cloning outcome. A technical report by Promega indicates that 3.1 kb fragment was clonable (link is attached) into pGEM®-T vector series. Do you have any experience in cloning large inserts, such as ~6 kb, into pGEM®-T Easy Vector?

2. What could be the screening strategy for positive clones? It seems that colony PCR by amplifying larger fragments is not a good approach.

3. What shall I do for the fifth isoformi (X5) that produces 2 bands (even under a gradient PCR of temperature from 58 to 68 C)?

https://www.promega.jp/resources/pubhub/enotes/comparing-cloning-efficiency-of-pgemt-and-pgemt-easy-vectors-to-topo-ta-cloning-vectors/?activeTab=0

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