Jeff:LabNotes/Microbiome/2012-5-11: Difference between revisions

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>Jgole
(Created page with "== '''Library Construction: Microwell Amplicons ''' == *Extracted 2 e coli amplicons and 2 microbiome ampliconsin 1 uL water *HMW buffer in tagmentation *Use Pol1 +ALS/NS tre...")
 
>Jgole
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== '''Library Construction: Microwell Amplicons ''' ==


*Extracted 2 e coli amplicons and 2 microbiome ampliconsin 1 uL water
*HMW buffer in tagmentation
*Use Pol1 +ALS/NS treatment
'''Pol 1 treatment'''
*Start with 1 uL DNA
*Add 1.5 uL ALS, 3min
*Put on ice and add 1.5 uL NS
Template  4 uL   
10x buffer 2 uL   
dNTP 1 uL         
N6(unmodified) 5 uL 
water 7 uL       
Pol 1 1 uL       
----------------------------------
Total 20 uL       
*37 C for 1 hr, 65C for 10 min
*Standard EtOH (Add 200 ng tRNA carrier) and elute in 7 uL water
'''Tagmentation'''
DNA in water  7 uL
HMW buffer  2 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
'''1st PCR round'''
DNA  13 uL
KAPA Robust mix  25 uL
Primer cocktail  1 uL
1 uM barcode (25-28)  1 uL
water  9.6 uL
50x SYBR .4 uL
--------------------------
Total  50 uL
*95 C 30s (95 C 10s, 62C 15s, 72 C 2 min)x20
'''Bead purfication'''
*Add 50 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'''
*Looks good
*HMW worked well
*Both e coli and microbiome samples amplified
*Will continue to process HMW samples (2 e coli and 2 microbiome)
*Test to see if there is still human contamination in microbiome samples
[[File:5-18-12 pcr.jpeg|400px]][[File:5-18-12 pcrgel.jpeg|400px]]

Latest revision as of 17:24, 21 May 2012