Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-2-27
Jump to navigation
Jump to search
Nextera Lib Construction Test on Sea Bac (CH-S-A)[edit]
Background[edit]
- I have already made Illumina libraries from the four highest concentrated samples on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/SeaMicrobiome/Calendar/2015/2015-2-18>. These four samples will also be used in a comparison test with two Nextera enzymes (original Nextera and supermutant):
Concentration CH-S-A 63.2ng/ul CH-S-B 69.2 CH-S-C 83.9 CH-S-D 35.2
- We want to dilute the samples to ~1ng/ul using the following dilutions:
Concentration ul Sample ul Water CH-S-A 63.2ng/ul 1 62 <- We will be using only this sample for today's test CH-S-B 69.2 1 68 CH-S-C 83.9 1 82 CH-S-D 35.2 1 34
- These libraries will be tested using the protocol provided by Andrew on <http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Protocols/tn5059>. The necessary reagents can be found in one of three places:
- Andrew's 4C box
- -20C 2nd shelf in Midas -20C box
- -20C 2nd shelf in Nextera primer box
- The Nextera supermutants are provided in the 50ml tubes labeled Control (the thick handwriting is 25x diluted enzyme)
- For this test, we'll need to run several reactions: different dilutions of the enzyme (up to 1x dilution and below) and also include a control tube with the original Nextera enzyme (already diluted 1/50)
- Below are the reactions and conditions we'll be testing today:
Rxn # Template Enzyme Enzyme Dil 1 CH-S-A (1ng) Nxt Old 1/50 2 "" Nxt SM 1x 3 "" "" 0.75x 4 "" "" 0.50x 5 "" "" 0.25x 6 "" "" 0.1x 7 Water (NTC) Nxt Old 1/50 8 "" Nxt SM 1x
Procedure[edit]
Prepare Enzyme Dilutions[edit]
- We want to dilute the supermutant to the following dilutions (from the original 25x concentration):
1x 1ul supermutant + 11.5ul UV treated TE buff + 12.5ul glycerol 0.75x 3ul 1x dilution + 1ul Water 0.50x 2ul 0.75x dilution + 1ul Water 0.25x 1ul 0.50x dilution + 1ul Water 0.1x 1ul 1x dilution + 9ul Water
- We also want to dilute ProtQ 1/100 from 20mg/ml. In order to do this, we add 0.2ul ProtQ + 200ul Water
Protocol[edit]
- Prep tagmentation mastermix (will do 8 reactions as described above)
1x 8.5x (ul) Template 1 -- Nxt 1 8.5 HMW Buff 1 8.5 Water 2 17
- 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 8.5x (ul) 10U/ul Exo- 0.1 0.85 25mM dNTP 0.4 3.4 Water 0.5 4.25
- Incubate: 37C 15min -> 75C 20min -> 10C forever
- Add the following i5 adaptor (N/S5xx, containing index 2) and 1ul i7 adaptor (N7xx, containing index 1) in the following order. This is based off of the Nextera protocol recommendations for low plexity reactions found on File:Cw 2015 02 26 Nextera DNA Sample Prep Guide.pdf
rxn # Indx 1 (i7) Indx 2 (i5) 1 N701 (TAAGGCGA) N501 (TAGATCGC) 2 N702 (CGTACTAG) N502 (CTCTCTAT) 3 N703 (AGGCAGAA) N503 (TATCCTCT) 4 N704 (TCCTGAGC) N504 (AGAGTAGA) 5 N705 (GGACTCCT) N505 (GTAAGGAG) 6 N706 (TAGGCATG) N506 (ACTGCATA) 7 N707 (CTCTCTAC) N507 (AAGGAGTA) 8 N708 (CAGAGAGG) N508 (CTAAGCCT)
- Prep PCR mastermix -> add 11ul to each rxn
1x 8.5x KAPA SYBR Fast (2x) 10 85 Water 1 8.5
- Incubate: 72C 3min -> [98C 10sec -> 63C 30sec -> 72C 3min]x13 -> 10C
- Purify using Ampure beads at 0.8:1 bead:sample ratio. Elute in 20ul H2O
- Run 3ul on TBE gel
- Purify again (same ratio) before sequencing if low-weight fragments appear on gel
Results[edit]
- Below is the gel image for the experiment: (Low Mass Ladder, x, rxn's 1->8)
File:Cw ZhangLab 2 2015-02-27 19hr 09min Nextera Lib Construction Test.jpg