The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II.
For 60bp run, we can use the library size of >180bp
For 80bp runs, the lower limit for the sequencing libraries should be 200bp.
If the library size is too small, some of the 80bp reads will reach to the other end of the adaptor sequences. we don't want to waste the sequencing $$ on the regions we don't want
The total size of the adaptors is 105bp, and there is another ~25bp of the capturing arm
The upper limit should be ~50bp above the lower limit
Fragmentation and end-polishing (Make blunt ends with 5'P)[edit]
The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
End-Repair Reactions
Fragmented DNA
85ul
10X End Repair Bufer
10ul
End Repair Enzyme Mix
5ul
Incubate tubes at RT for 30minutes.
Perform a Qiaquick purification and elute with 39ul EB buffer.
NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
A-Tailing Reactions
Blunet-end DNA
37ul
10X dA-Tailing Reaction Buffer
5ul
Klenow Fragment (3'-5' exo-)
3ul
H2O
5ul
Size selection using Invitrogen 2% SizeSelect gel[edit]
Fill any unused well with 30ul EB Buffer
Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube
Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well
Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.
Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).
Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.[edit]
Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
ul
End-repaired & size selected DNA
36
40uM adaptor2
2
5X Quick Ligase Buffer
10
Quick Ligase
2
Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB.