Friday 9/18/15

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.

Adaptor 1
11x
100 um 5ILL-NR
.6 uL
100 um Anti-ILL
.6 uL
NFW
58.8 uL
Adaptor 2
11x
100um 3ILL -NR
.6 uL
100um Anti-ILL
.6 uL
NFW
58.8 uL
Ligation reaction master mix
1x
11x
2 uM Adaptor 1
5 uL
55
1 uM Adaptor 2
5 uL
55
T4 ligase
1 uL
11
T4 ligase buffer with 10 mM ATP
4 uL
44
NFW
25
275

*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.
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2 thoughts on “Friday 9/18/15

  1. 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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