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Anorexia anx/anx mutant mice (view source)
Revision as of 14:56, 29 November 2024
, 14:56, 29 November 2024added range of concentrations so far
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+
−
Measure DNA quantity/quality with Nanodrop.
Measure DNA quantity/quality with Nanodrop.
−* yield was xxx - xxx ng/ul genomic DNA.
+* yield was 60 - 1100 ng/ul genomic DNA.
===PCR===
===PCR===
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For 50 ul reaction:
For 50 ul reaction:
−* xx ul Gotaq
+* 25 ul Gotaq
−* xx ul mixture of 5 uM forward/reverse Tyro3 primers
+* 10 ul mixture of 5 uM forward/reverse Tyro3 primers
−* xx ul dH<sub>2</sub>O
+* 14 ul dH<sub>2</sub>O
* 1 ul of genomic DNA
* 1 ul of genomic DNA
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PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C).
PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C).
−====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 ul of SYBR safe 10000x while cooling). Make mini-gel with 12-tooth comb
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load 10 ul of 50bp ladder; 10 ul of PCR product + 2 ul purple loading dye
load 10 ul of 50bp ladder; 10 ul 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 500bp [CHECK]
+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 dH<sub>2</sub>O [ because worried about pH of dH<sub>2</sub>O, 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 ul in dH<sub>2</sub>O [ because worried about pH of dH<sub>2</sub>O, now trying kit elution buffer which I think is Tris-Cl pH 8.5]
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Measure DNA quantity/quality with Nanodrop.
Measure DNA quantity/quality with Nanodrop.
−* yield was xxx - xxx ng/ul clean PCR product
+* yield was 20 - 65 ng/ul clean PCR product
===Re-Amplification===
===Re-Amplification===
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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, 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.