ࡱ> 241c Mbjbj** . HSbHSb R44444HHH8H0($~d=4=44RWWWF44WWWWPdaRWh0WK$W4WW==oB : DNA purification on AMPure XP beads This method is standard for purification of DNA libraries and intermediates during preparation of Illumina libraries. This protocol is basically as recommended by NEB and works in the NEBNext pipelines as well as custom procedures. Please read the notes before using this method as it will save misery later! Protocol Let an aliquot of beads come to room temperature for 15-30 min before use Beads can sit on bench for a few hours but remember to put away afterwards. AMPure beads cost > 10 / ml and we dont recommend using beads that have been left out overnight (we dont know if it is a problem but dont risk it) Vortex very well until the brown pellet at the bottom of the tube is resuspended Add bead solution to the sample for cleaning at the given ratio Mix well by pipetting or vortexing and incubate for 5 minutes at room temperature Meanwhile make up 450 l 80% ethanol per sample this must be used within a few hours. I have a 15ml tube set aside for this. Place tube on a magnet and allow solution to clear (3-5 minutes) Remove and discard the solution, leaving the beads in the tube While the tube is still on the magnet, add 200 l 80% ethanol After a few seconds up to a few minutes, remove the ethanol wash with a P200 Repeat the wash with another 200 l 80% ethanol Use a P10 or P20 with a P2/10 tip to remove any residual ethanol at the bottom of the tube (there is almost always some) Leave the tube open on the magnet for 5 minutes to dry (not more than 10 minutes) Remove tube from magnet and add the required elution volume of 0.1x TE Vortex and leave on bench for 2 minutes Put tube back on magnet, let clear and pipette the eluted material to a new tube You cannot retrieve all the material. Leaving 2 l is easy, 1 l is what we normally leave, 0.5 l is achievable but hard Discard the beads Notes On receipt of the bottle of AMPure beads, we split these into 1ml aliquots and store at 4. The beads work long after the Use by date in our hands. AMPure clean-ups begin by mixing the bead solution and the material to be cleaned at a defined ratio. It is a common misconception that the important thing is the amount of magnetic beads added. This is not true. Rather, the AMPure buffer contains PEG and salt, and the final concentration of PEG and salt in the mixture defines the size range of DNA fragments that will bind. If the material to be cleaned already contains PEG or a lot of salt this will affect the binding, so if cleaning e.g.: a ligation reaction for which PEG is often present in the buffer then you need to be aware of this. Various images are available showing bead clean-ups to give an idea what effect changing the ratio between beads and reaction volume has, irritatingly they are quite variable. In our hands 1:1 has a size cut off around 100 bp. We purify our amplified NEBNext libraries twice at 0.9x which works very well to remove adaptor dimers or 0.8x if there are a lot of adaptors although this will start to cut into the low end of the library      FILENAME AMPure clean-up v1.0 Houseley lab  PAGE 2 #$% " R S Y [ c d w x ) * #M()TV\]  #FGݿݿݿٵٵٵٵݱݭݭݭݱݩjh&RU h)h)h)huIh59h[p!OJQJ^JhmGOJQJ^JhmGhw6hmGhmG6h[p!hmGhwhMh)5hM5CJ(aJ(hw5CJ(aJ(<$%Z [ d e % & x y : ; z {   8 ^gdmGgd)$a$gd)8 9 MNvwBCU\]FGgdmGgd[p!^gd[p! #$2689:;<BCDEGHIJLMͺ h)h)jh(0JU*huI0JmHnHu* h(0Jjh(0JUh%huImHnHuh(jh(UhuIjh&RUh&R789:FGHIJKLM,1h. A!"#$% s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< M #  >@MOQTM 8 M )AHJT!8@0(  B S  ?     K N $'     K N 3RS[[ "'(.BCT]   # E  6 8 ; E G K N RS[[ "'(.BCT]   # E N %$Mmp.&R[p!%*Mr2; BmGcH7TYs,[QycRdgxomu./|fL0(sFuw7uIbRn(?)d59 @****M @UnknownG.[x Times New Roman5Symbol3. .[x Arial7..{$ CalibriA$BCambria Math"1hÇÇ'1 1 !4 2qHP ?72!xx?mT  Prepare 0jhousele Jon Houseley Oh+'0|  8 D P\dlt Prepare 0 jhouseleNormalJon Houseley5Microsoft Office Word@r@ZȲa@a1  ՜.+,0 hp  University of Edinburgh   Prepare 0 Title  "#$%&'(*+,-./03Root Entry F֨a51TableWordDocument. SummaryInformation(!DocumentSummaryInformation8)CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q