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. Author manuscript; available in PMC: 2023 Jul 13.
Published in final edited form as: Nat Protoc. 2022 Nov 16;18(2):396–423. doi: 10.1038/s41596-022-00766-8

Fig. 6: Gel images showing expected products from Stage II of the protocol.

Fig. 6:

Abbreviations are as in Fig. 3. a, An example final gel image including the products of the MRP, PAL, PTC and TEV reactions from the production of a 30-aa library. The MRP product appears as a single band of mRNA. The PAL reaction material appears as two bands, an upper band for the adapter-ligated mRNA (mRNA+PuroAdapter) and a lower band of unligated mRNA (MRP product). The PTC reaction material runs as three dominant bands: the upper band is the adapter-ligated mRNA with the translated peptide attached (mRNA+PuroAdapter+Peptide), the middle band is the adapter-ligated mRNA (with no peptide) and the lower band is unligated mRNA. As a result of RNase treatment, the unligated mRNA product is not observed after the TEV reaction, but the puromycin-ligated cDNA and puromycin-ligated cDNA bound to peptide are both present. The faint band seen between the mRNA and mRNA+PuroAdapter in the PTC and below the cDNA+PuroAdapter band in the TEV lanes is a byproduct formed during in vitro translation. This is a 6% TBU gel run for 45 min in heated 1× TBE buffer. b, An example image of a quality-control gel run during the production of a 30-aa library. The single lower band in the PAL reaction material is the unligated puromycin adapter (PuroAdapter). It is also common to see a band near the bottom of the gel in the PTC lane, which is probably the capture oligo. This is a 10% TBU gel run for 75 min in unheated 1× TBE buffer.