Ligation of adaptors to digested samples.
Need to make new adaptors each time. Recipe below is for 10 samples (+1 for pipet error) to make adaptors at 2 uM. Combine and let sit for 10 minutes at room temp – I made sure stocks were completely melted.
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Adaptor 1
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11x
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100 um 5ILL-NR
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.6 uL
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100 um Anti-ILL
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.6 uL
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NFW
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58.8 uL
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Adaptor 2
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11x
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100um 3ILL -NR
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.6 uL
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100um Anti-ILL
|
.6 uL
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NFW
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58.8 uL
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Ligation reaction master mix
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1x
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11x
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|
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2 uM Adaptor 1
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5 uL
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55
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1 uM Adaptor 2
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5 uL
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55
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T4 ligase
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1 uL
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11
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T4 ligase buffer with 10 mM ATP
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4 uL
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44
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NFW
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25
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275
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*Note: the Meyer protocol calls for 1.0 uL rATP and 4.0 uL T4 ligase buffer. My buffer had 10 mM rATP included, so I just used 25 uL of NFW instead of 24uL and did not add more rATP.
40 uL master mix with 10 uL of digested DNA. Held at 16degC for 2 hours and 15 minutes. Put in -20C.
Getting My Genes Wet 
Just out of curiosity, why did you deviate from the Meyer protocol? I’m sure they know that the T4 Ligase Buffer already contains ATP, so it seems like they need/want additional ATP in the ligation reaction.
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The Meyer protocol is very specific about reagents. When I saw that 10 mM ATP was listed on the front of the buffer, I assumed (maybe incorrectly) that that was a recent change to NEB’s buffer. You have a point though, I’ll order some NEB ATP and follow the protocol as listed.
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