Changes

PCR master-mix: [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega Gotaq colorless master mix].  

PCR master-mix: [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega Gotaq colorless master mix].  

For 50 ul reaction:

For 50 λ reaction:

* 25 ul Gotaq

* 25 λ Gotaq

* 10 ul mixture of 5 uM forward/reverse Tyro3 primers

* 10 λ mixture of 5 uM forward/reverse Tyro3 primers

* 14 ul dH₂O

* 14 λ dH₂O

* 1 ul of genomic DNA

* 1 λ of genomic DNA

====Gel====

====Gel====

2% agarose in TAE (2 g agarose in 100 ml TAE, microwave 2 min at 50% power; add 10 ul of SYBR safe 10000x while cooling). Make mini-gel with 12-tooth comb

2% agarose in TAE (2 g agarose in 100 ml TAE, microwave 2 min at 50% power; add 10 λ of SYBR safe 10000x while cooling). Make mini-gel with 12-tooth comb

load 10 ul of 50bp ladder; 10 ul of PCR product + 2 ul purple loading dye

load 10 λ of 50bp ladder; 10 λ of PCR product + 2 ul purple loading dye

run at 90V for 30 min (or 65V for 60 min for sharper bands). Red dye front migrates at about 370bp, similar to bromophenol blue.

run at 90V for 30 min (or 65V for 60 min for sharper bands). Red dye front migrates at about 370bp, similar to bromophenol blue.

====PCR clean-up====

====PCR clean-up====

[https://omegabiotek.com/product/pcr-clean-up-kit-e-z-n-a-cycle-pure/ E.Z.N.A. Cycle Pure Kit (V-spin)] yields 30 ul in dH₂O [ because worried about pH of dH₂O, now trying kit elution buffer which I think is Tris-Cl pH 8.5]

[https://omegabiotek.com/product/pcr-clean-up-kit-e-z-n-a-cycle-pure/ E.Z.N.A. Cycle Pure Kit (V-spin)] yields 30 λ in dH₂O [ because worried about pH of dH₂O, now trying kit elution buffer which I think is Tris-Cl pH 8.5]

Measure DNA quantity/quality with Nanodrop.

Measure DNA quantity/quality with Nanodrop.

* yield was 20 - 65 ng/ul clean PCR product

* yield was 20 - 65 ng/λ clean PCR product

===Re-Amplification===

===Re-Amplification===

To get higher concentration of DNA for digest, repeat PCR using 1 ul of clean PCR product and clean up.

To get higher concentration of DNA for digest, repeat PCR using 1 λ of clean PCR product and clean up.

* yield was xxx - xxx ng/ul clean re-amplified PCR product

* yield was xxx - xxx ng/λ clean re-amplified PCR product

(could also pool repeated original PCR Rxns from genomic DNA, but that seems inefficient. Re-amplifying risks PCR artifacts/mutations, though ...)

(could also pool repeated original PCR Rxns from genomic DNA, but that seems inefficient. Re-amplifying risks PCR artifacts/mutations, though ...)

===Digestion===

===Digestion===

* 1 ul [https://www.neb.com/en-us/products/r0126-nlaiv NlaIV] (2 units)

* 1 λ [https://www.neb.com/en-us/products/r0126-nlaiv NlaIV] (2 units)

* 5 ul [https://www.neb.com/en-us/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/restriction-endonucleases/cutsmart rCutSMart Buffer 10x]

* 5 λ [https://www.neb.com/en-us/products/restriction-endonucleases/hf-nicking-master-mix-time-saver-other/restriction-endonucleases/cutsmart rCutSMart Buffer 10x]

* 1 ug clean Tyro 3 PCR product

* 1 ug clean Tyro 3 PCR product

* H₂O to 50 ul

* H₂O to 50 λ

Incubate at 37 C for 1 hour, heat inactivate 65 C for 20 min.

Incubate at 37 C for 1 hour, heat inactivate 65 C for 20 min.

NEBiolabs says to mix digest with purple dye with 0.5% SDS to separate protein from DNA. SDS may interfere with SYBR Safe if stain mixed in the gel, so may need to post-stain the gel.

NEBiolabs says to mix digest with purple dye with 0.5% SDS to separate protein from DNA. SDS may interfere with SYBR Safe if stain mixed in the gel, so may need to post-stain the gel.