Jeff:LabNotes/Microbiome/2011-12-8
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In Situ Tagmentatation[edit]
- I believe that doing in situ tagmentation will help with contamination problem
- The amplicons will be tagged prior to extraction, and if all the transposases are used up, there is no room for contamination in this step
- I also want to see if protease helps
Tagmentation
DNA in water 6.5 uL HMW buffer 2 uL 1:10 diluted enzyme 2 uL 50x SYBR green .5 uL ---------------------- Total 10 uL
- Pipette 10 uL mix onto array
- 55C for 5 min
- Look under microscope
- For protease, the library was added to 5 uL water, 1 uL 1:500 protease and subjected to standard protease temps
- 2 amplicons were extracted due to evaporation issues, and 1 for protease, and an NTC
1st PCR round
DNA 6 uL BST 1 uL KAPA fast mix 15 uL 10 uM orange primer .24 uL 10 uM blue primer .24 uL water 7.52 uL -------------------------- Total 30 uL
- 60 C 20 min, 72 C 3 min, 95 C 30s (95 C 10s, 58 C 30s, 72 C 2 min)x20, 72C 3 min
Results
- The amplicons shot up meaning that tagmentation worked.
- Protease seemed to inhibit PCR
- The gel looks better, but still not right. There is a smear, but it starts about 100 bp too low. This is most likely because it took some time to remove the amplicons and the transposases were still active.
- There is still a band need to figure this out
File:12-8-11 npcr.jpeg File:12-8-11 gel.jpeg
Microwell MDA with N6[edit]
Put everything together.
- Have successfully performed MDA cleanly in microwells with all sub parts amplifying cleanly.
- Grew fresh E coli and washed 2x in UV'd, filtered PBS
- I found that the contamination is most likely coming from the post amplification steps, specifically unclean clean pipette tips during extraction and ethanol precipitation
- These can be controlled: omit ethanol precip (found that this step is not necessary) and UV tips after a clean breaking procedure
- Recently made new clean PDMS at half thickness. These should be easier to heat and much cleaner than the ones before.
Preparation
- View microwells under microscope. Make sure there is limited debris in the wells. Also check under GFP filter to ensure there are no prior large DNA fragments
- Apply ethanol to array. Let sit for 10 min
- Apply vacuum to array for 30 s so the ethanol penetrates the wells.
- UV clean water and microwell array for 10 min
- Wash out the ethanol with clean water several times. Then add water and put on shaker for 10 min at low shaking speed. Transfer out of the hood in a clean (UV'd/ethanol/DNAway) pipette tip box
- Vacuum for 30 s.
- Remove the water by blowing with air from chemical hood.
- Add 1% BSA to well and UV for 5 min. Then let the BSA soak for 1 hr.
- We are now ready to move onto MDA
MDA
- UV everything needed in reaction that does not contain DNA or enzymes (water, BSA, buffers, etc). Also UV/DNAway hood, pipettes, tweezers, microscope stage insert and cover, etc.
- Obtain a flat dry ice brick (or put ice block in -80 C for 30 min). Place on a styrofoam lid and UV for 10 min. Also UV the metal thermocycler slide holder for 10 min and wipe with DNAway. UV slide stage adaptor as well.
- Dilute cells in PBS to 20 cell/uL. Make the following mix. 22uL water, 6 uL diluted cells, and 2 uL BSA.
- UV/ozone treat, transporting in clean pipette box. 5 min prime and 3 min treatment.
- UV array for 5 min
- Spread 5 uL cells evenly over array
- Clean vacuum with EtOH and DNAway. Vacuum seed for 30 s.
- Perform freeze/thaw lysis. Place array on dry ice with tweezers for 1 min. Remove and place on slide holder for 1 min. Repeat 2x.
- Add 5 uL ALS, wait 3 min, then 5 uL NS.
- Create the master mix: 5.8 uL water, 5 uL 200 uM N9 primer, .6 uL 50x Sybr, .8 uL dNTP, 3.4 uL buffer, 1 uL phi29.
- Cover with clean mineral oil
- 30 C for 2 hr, 65 C 20 min, 4 C forever