were used for 2nd-strand cDNA synthesis.

Hi,

I am entirely frustrated and thought I'd appeal to this community for help-
I have amplified mRNA from a prokaryotic community and have microgram amounts of decent mRNA. I then used Invitrogen's double-stranded cDNA synthesis kit which ends in spiking the reaction with T4 polymerase so as to render the cDNA ends blunt. I am having absolutely no luck getting any clones when trying to clone into a blunt vector. All the cloning controls work as expected and the transformation efficiency of the comp. cells is fine, but I can't get any clones with my cDNA. I'm starting to wonder if there's a problem with the cDNA quality, but how can I assess quality? It looks like a shmear on a gel (which is expected since it's all sizes, up to ~1.5 Kb) and the concentration is reasonable. Why won't it clone??? Suggestions?

Thanks,
Rachel

I then used Invitrogen's double-stranded cDNA synthesis kit ..

The SuperScript Double-Stranded cDNA Synthesis Kit ..

Protocol for NEBNext® Ultra™ II Non-Directional RNA …

Howdy all,

I'm generating cDNA from purified poly(A)RNA, following a protocol and noticed that there is no second strand primer. AND, the thermocycling conditions for the second strand are fairly cold - 2hrs @ 16C. Is it possible to have cDNA synthesis occur spontaneously without a primer? I'm wondering if this is a missing element in my protocol; the first strand primer (a polyT primer) shouldn't match the first strand synthesized, so what starts the second strand??? And what does the DTT do?

Thanks for any info!

Here's the scheme - most reagents are supplied by Invitrogen:

First Strand Synth -

polyA RNA
dT (polyT) primer
dNTPs
water


first strand buffer
DTT (what the heck is that?? comes with the superscript kit)
RNAseOUT
SuperScript III RT



Second Strand Synth
second strand buffer
dNTP
DNA ligase
DNA polymerase
RNase H
water

T4 DNA polymerase

EDTA to stop rxn.

how can we design primer to synthesis second strand of ..

ProtoScript II – NEB ProtoScript II First Strand cDNA Synthesis Kit and random primer mix; SuperScript II – Invitrogen SuperScript First Strand Synthesis System for RT-PCR and random hexamers; SuperScript® III – Invitrogen SuperScript III First Strand Synthesis SuperMix and random hexamers. The following reagents are supplied with this product:

DNA⁄RNA Modifying Enzyme Buffers
The following mixture was added to the purified cDNA for second strand synthesis: 1 μl ..

Protocols for Reverse Transcription (cDNA Synthesis) ..

Synthesis of the second strand of cDNA using pol ∝-primase is provided

Web, Imagens e Vídeo · A Completa Pesquisa Geral

(NEB #M0277) First Strand cDNA Synthesis (No-RT Negative Control Reaction) (NEB #M0277)

Is it possible to have cDNA synthesis occur spontaneously ..