Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-31: Difference between revisions
Jump to navigation
Jump to search
>Cjwei No edit summary |
>Cjwei |
||
Line 51: | Line 51: | ||
Water 0.5 4.75 | Water 0.5 4.75 | ||
*Incubate: 37C 15min -> 75C 20min -> 10C forever | *Incubate: 37C 15min -> 75C 20min -> 10C forever | ||
Revision as of 17:41, 31 March 2015
Nextera Lib Construction on Catalina Harbor Samples
Background
- Previously, I tested the supermutant Tn5 on samples CH-S-A/B/C/D and found that the 0.25x concentration worked the best for 1ng of input (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-3-10>
- Today, I'll be performing library construction using the supermutant Tn5 on the remaining Catalina Harbor samples, which were all >100ng total yield.
- We want to dilute the samples to ~1ng/ul using the following dilutions:
Concentration ul Sample ul Water Test 1 5.22ng/ul 1 4.22 Test 2 5.09 1 4.09 CH-P-A 6.67 1 5.67 CH-P-B 18.8 1 17.8 CH-P-C 14.1 1 13.1 CH-P-D 13.6 1 12.6 CH-O-A (Filter 1) 12.6 1 11.6 CH-O-B (Filter 2) 27.6 1 26.6
- The necessary reagents can be found in one of four places:
- Andrew's 4C box
- -20C 2nd shelf in Midas -20C box
- -20C 2nd shelf in Nextera primer box
- -20C 3rd shelf Brandon's Tn5 box (contains the EZ-Tn5 and supermutant)
- Below are the reactions we'll be using today:
Rxn # Template Enzyme Enzyme Dilution 1 Test 1 sm-Tn5 0.25x 2 Test 2 "" "" 3 CH-P-A "" "" 4 CH-P-B "" "" 5 CH-P-C "" "" 6 CH-P-D "" "" 7 CH-O-A "" "" 8 CH-O-B "" "" 9 --(NTC) "" ""
Procedure
Prepare Enzyme Dilutions
- We want to dilute the supermutant-Tn5
- supermutant-Tn5 provided in 25x concentration, so dilute using the following:
0.5ulA + 0.5ulB + 99ul dilution buffer
- We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 2ul ProtQ + 198ul Water
Protocol
- Prep tagmentation mastermix
1x 9.5x (ul) Template 1 -- Nxt 1 9.5 <- supermutant Tn5 HMW Buff 1 9.5 Water 2 19
- Incubate: 55C 5min -> remove tubes and put on ice block
- Add 1 ul 1/100d ProtQ to each reaction
- Incubate: 50C 10min -> 70C 20min -> 10C forever
- Prep Exo- mastermix (add 1ul to each rxn)
1x 9.5x (ul) 10U/ul Exo- 0.1 .95 25mM dNTP 0.4 3.8 Water 0.5 4.75
- Incubate: 37C 15min -> 75C 20min -> 10C forever