AlanFung:LabNotes/Probe/2012-4-4

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Probe Synthesis CES36K 18BP[edit]

Expansion PCR[edit]

Content Volume X2.1
CES36K18BP (20nM) 1 2.1
2X iQ MasterMix 50 105
100uM AP1V6 0.4 0.84
100uM AP2V6 0.4 0.84
H2O 48.2 101.22
 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 15 -> 72C 3min -> 15C hold  
 Purify the amplicons with two Qiaquick columns, eluted with 30ul EB.
 Yield: 10.3ng/ul x 60ul = 618ng
 Concentration = 10.3ng/ul / (140 * 660) = 108nM
 Take 20ul, add 196ul EB -> 10nM

Production PCR[edit]

Content Volume X33
10nM 1st round amplicon 0.2 6.6
2X Kapa SYBR qPCR Master Mix 50 1650
100uM pAP1V61U 0.4 13.2
100uM AP2V6 0.4 13.2
H2O 44 1452

95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold.

  • I did a test run with one well and see how many cycles i need to run

File:120420.JPG

  • Looks like 12 cycles will be enough
  • Now I will go ahead and move on with the production
Content Volume needed Volume
100% ETOH 2.5x sample 8000
3M NaOAC 0.1x sample 320
Glycoblue 1/300x sample 10.66666667
sample sample volume 3200
  • Mix all content by repeat pipetting using multichannel pipette
  • Pool all 24 wells PCR product into tray using multichannel pipette(P300)
  • Mix all content by repeat pipetting using multichannel pipette
  • Aliquot about 3750uL to 2 x 15mL tube
  • Store in -80C for more than 20 mins or forever
  • Continue on Apr29 [[1]]

Production PCR[edit]

  • Continue from Apr04 [[2]]
  • Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
  • Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C
  • Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
  • Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
  • Centrifuge at 10,000rpm for 5min at 4C
  • Remove all liquid, air dry sample for 5min or until samples dried out
  • Resuspend the DNA with 100uL RNAse free H2O
  • Re-purify using 4 qiaquick tubes elute with 41ul EB
  • Yield 4x40x201ng/ul=32.16ug
  • Store in 4C, continue on Apr30 [[3]]

Nanodrop[edit]

Lambda exo digestion[edit]

  • Split the libraries into half, 80ul each around 16.8ug
Content Volume
Qiaquick amplified amplicon 80
10X lambda Exo Buffer 10
Lambda Exo 10
H2O 0
Total 100
  • Incubate at 37C for one hour
  • Purify with 4xQiaquick elute with 31ul EB (since each qiaquick column max binding capacity is 10ug)
  • Yield ssDNA: 75.5ng/ul x 120ul = 9.06ug
  • Labeled tube and stored @ -20C
  • Continue on 5/1/2012 [[4]]

Removal of amplification adaptors[edit]

  • To 75ul ss-amplicon (5.7ug) , add 5ul USER enzyme, 37C 1h;
  • Add 10ul 10x Dpn II buffer, 5ul 100uM RE-DpnII-V6 guide oligo, 94C 2min -> 37C 3min -> add 5ul Dpn II -> 37C 2h
  • add 5ul USER, 10ul 10x buffer, 85ul H2O
  • -> 37C 2h -> 75C 20min -> purify with two Qiaquick column
  • Continue May 2 [[5]]

Gel Quantification[edit]

  • Elute with 120ul EB and used 1ul to run gel

File:ZhangLab 2 2012-05-02 16hr 03min.jpg

  • Digestion didn't complete
  • Talked to Noi and she thinks so too
  • Will work together with Noi on 5/7 and repeat the digestion protocol.

Troubleshooting May7[edit]

  • Noi and I think that the probes are not digested completely

USER Digestion[edit]

  • from the 75.5ng/ul tube, take 40ul product add 2.5ul USER enzyme
  • Incubate at 37C for 1.5hr and run a denaturing gel
  • Since I ran a TBE gel on my end product from last week, I will rerun on a denaturing gel so that the 2nd structure wont be the reason why we see the larger than expected DNA size

File:ZhangLab 2 2012-05-07 13hr 31min.jpg

  • Turns out the end product is at the expected size, it was the TBE gel leading to larger than expected DNA size.
  • Will move on with the tube we started today and pool it with the end product and perform a size selection for both tubes.

Page Denaturing Gel[edit]

  • ssDNA nanodrop result:28.8ng/ul x 150ul = 4.320ug
  • Limit for 2D gel is around 1ug I will run 4 gels
  • Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel
  • Load 4 gels into the gel running module
  • Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
  • Pre-run the gel for 30 min at 200V
  • Dilute sample to no more than 40ng/uL
  • Dilute TBE-Urea sample buffer (2X) to 1X in each tube.
For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer 
  • Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins
  • Flush the wells very well with P1000 & P200 pipette to remove all urea.
NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP 
  • Load the samples into the wells, run at 200V for 30 min.
  • Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
  • Place tray on an orbital shaker for ~5 min.

Purify the product from the gel[edit]

  • Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
  • Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
  • Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
  • Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
  • Spin down all tubes at 13,000rpm for 2min
  • In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.

Quantification of DNA using denaturing gel and a quantitative ladder[edit]

Combine all tubes into a 1.5mL tube

1. Add 1ul of low mass ladder, 4ul of H2O and 5 uL of 2x TBE-Urea buffer
1. Add 2ul of low mass ladder, 3ul of H2O and 5 uL of 2x TBE-Urea buffer
1. Add 4ul of low mass ladder, 1ul of H2O and 5 uL of 2x TBE-Urea buffer
2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer
2. Dilute sample 10x (2uL sample + 8 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer
3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min.
4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea.
5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min.  Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL.
6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.

File:ZhangLab 2 2012-05-14 15hr 46min.jpg

  • Using the linear trendline we obtain the following formula, where x is ng and y is the intensity
  • y = 546.85x + 2758.3
  • the 1ul probe's intensity is at 18876.28 which converts to 29.47ng
  • the 2ul probe's intensity is at 22667.29 which converts to 36.41ng