Protocol for In Vitro Transcription (IVT) DNA Template Construction Kamen P. Simeonov, Hirdesh Uppal Regarding – DOI: 10.1371/journal.pone.0100134
Have ready before starting: DNA Ladder for gel SYBR E-Gel ORF Primers @ 10uM ORF Template DNA @ 3ng/uL 2x KAPA HiFi Mix QiaQuick Purification Kit Qiaquick Gel Extraction Kit 10x Ligation Buffer Ampligase UTRs @ 5uM Splints @ 2uM Template Primers @ 10uM Template Tail Primer @ 5uM Sequencing Primers for ORFs ORF PCR: Reaction Mix (25uL Total) 2x KAPA Mix: 12.5uL 10uM Forward Primer: 0.75uL 10uM Reverse Primer: 0.75uL 3ng/uL Template DNA: 1uL Water: 10uL Note: Primers are ORF specific PCR Setup 1 cycle: 95C 5min 20 cycles: 98C 20sec 65C 15sec 72C 60sec 1 cycle: 72C 5min 4C HOLD Purification: -Run a gel (Speed E-Gel protocol on 1.2% gel, load ~100ng in 20uL per well) to make sure the amplicons are the correct length, may need to extract the correct band using CloneWell Gel -Purify tubes with Qiaquick Purification Kit (if not doing gel band purification) -Nanodrop each tube (yield ~55ng/uL in 50uL) Splint Ligation: Reaction Mix (50uL Total) 10x Ligation Buffer: 5uL 5uM 5’UTR: 2uL 5uM 3’UTR: 2uL 2uM 5’Splint: 2.5uL 2uM 3’Splint: 2.5uL DNA from above: ~50ng total maximum (corresponds to ~40nM) – 1uL should be good Water: to 50uL Add 1 unit of vortexed Ampligase
Protocol for In Vitro Transcription (IVT) DNA Template Construction Kamen P. Simeonov, Hirdesh Uppal Regarding – DOI: 10.1371/journal.pone.0100134
Vortex mix thoroughly Note: Splints are ORF specific, but UTRs are not Ligation Setup 5 cycles: 95C 10sec 45C 1min 50C 1min 55C 1min 60C 1min Purification: -Purify both tubes with Qiaquick Purification Kit Large Scale Template Tail PCR: Reaction Mix for 6 reactions (153uL Total) 2x KAPA Mix: 76.3uL 10uM Forward Template Primer: 4.6uL 5uM Reverse Tail Template Primer: 9.2uL DNA: 31uL from ligation Water: 32uL Template Tail PCR Setup is same as ORF PCR (except with 30 cycles) Run some of the template PCR product next to the ORF PCR product on a 1.2% gel with Speed E-Gel protocol to see if there is a discreet band of correct length (slightly longer than ORF PCR product) Purification: -Template PCRs should be purified using traditional gel extraction to avoid loss of product -Run out one 25uL PCR per well on a 1.2% SYBR E-Gel -Each gel has 12 lanes, Hnf4a, Foxa1 and Foxa3, nGFPd can be grouped on 2 gels. -Hnf1a, Foxa2, and Gata4 will each take up an entire gel -Open gel with a razorblade and excise bands with a new blade for each template -Put excised bands for each template into their corresponding tube -Gel purify using the Qiaquick Gel Extraction Kit -Re-purify with Qiaquick PCR purification kit (elute with water) since gel purification produces consistently bad 260/280 ratio -Adjust concentration to rounded number -Sequence templates -Templates can now be directly used to template IVT
