Jeff:LabNotes/Microbiome/2012-8-23: Difference between revisions

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== ''Tagmentation test using Genomic DNA''' ==


*Test 4 samples, each with 25 ng genomic DNA starting template
*Sample 1: PC in water
*Sample 2: DNA is EtOH precipitated and eluted in water
*Sample 3: DNA in New prep below w/o Pol I.  ALS and NS premixed as to be neutral
*Sample 4: Pol I treated DNA using New prep
'''Pol 1 treatment'''
*Start with 1 uL DNA
*Add 1.5 uL ALS, 3min
*Put on ice and add 1.5 uL NS
'''New Prep'''
Template  4 uL   
10x buffer 4 .7 uL   
dNTP .35 uL         
N6(unmodified) .35 uL 
water .4 uL       
Pol I .5 uL       
----------------------------------
Total 7 uL       
     
*37 C for 30 min, 65C for 10 min
'''Tagmentation'''
DNA in water  7 uL
HMW buffer  2 uL, (.6 uL for New prep samples for a total of 2 uL)
water (for new prep only) 1.4 uL
1:50 diluted enzyme  1 uL
----------------------
Total 10 uL
*55C for 5 min
'''Protease treatment'''
*Add 1 uL 1:100 diluted protease
*10 min 50C,  20 min 70C
'''Klenow'''
*Add 1 uL exo- klenow and 1 uL dNTP to each
*37C for 15 min, 75 for 20 min
'''1st PCR round'''
DNA  13 uL
KAPA Robust mix  15 uL
Orange  .6 uL
blue  .6 uL
50x SYBR .24 uL
--------------------------
Total  30 uL
*95 C 1min (95 C 10s, 65C 15s, 72 C 45s)x8
'''Second round PCR'''
*Pause PCR program and add following to each
1st round PCR mix 30 uL
KAPA Robust mix 35 uL
Adapter 1 2 uL
Adapter 2 Barcode 2 uL
SYBR Green 1 .56 uL
water 30.44 uL
----------------------
Total 100 uL
1 min 95C, (95 C 10s, 65C 15s, 72 C 45s)x3, (95 C 10s, 72C 1 min)x4, 72C 3min
'''Bead purfication'''
*Add 100 uL beads to each, mix, and let sit at RT for 8min
*Transfer tubes to magnetic rack and let sit for 5 min
*Remove supernatant
*2x 80% EtOH wash
*Sit for 15 min
*Elute in 23 uL Qiagen buffer
'''Results'''
*There is a 1 cycle difference between PC and EtOH, implicating 1/2 the DNA is lost
*Even though the buffer for the Pol I reactions is the same as tagmentation buffer, tagmentation is very inefficient.  ALS is prepared as diluted KOH only (no DTT or EDTA).  Not sure what is happening.  Only differences are K and CL ions from ALS/NS, and dNTP's/N6 primer from Pol I mix
*When the Pol I reaction was run, it is entirely inhibited meaning the enzymes are somehow competing (even though Pol I is killed)
*In any case, EtOH continues to be the best option, and I will only use this in the future
[[File: 11-14-12 pcr.jpg|400px]]  [[File: 11-14-12 gel1.jpg|400px]]

Latest revision as of 21:16, 14 November 2012