Sam:LabNotes/Microbiome-new/2010-9-24: Difference between revisions

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>Sam Chiang
>Sam Chiang
Line 177: Line 177:
  94C 3min -> (94C 30sec -> 60C 30sec -> 72C 1min) x 30cycle -> 72C 5min -> 15C forever
  94C 3min -> (94C 30sec -> 60C 30sec -> 72C 1min) x 30cycle -> 72C 5min -> 15C forever
*Basically I use the standard PCR protocol with a longer extension time(1min)
*Basically I use the standard PCR protocol with a longer extension time(1min)
*10uL PCR products were evaluated using 2% agasrose E-gel(run 12min)
*10uL PCR products (leave 40uL for PCR product sequencing)were evaluated using 2% agasrose E-gel(run 12min)
  Results:  
  Results:  
   
   
  [[File:sam092810-PCR validation no1-no4.jpg|500px]] [[File:sam092810-PCR validation no5-no6.jpg|500px]]
  [[File:sam092810-PCR validation no1-no4.jpg|500px]] [[File:sam092810-PCR validation no5-no6.jpg|500px]]

Revision as of 05:50, 30 September 2010

Cloning Sanger sequencing test on E.coli gDNA MDA amplicons

Background

  • To optimize the Sanger sequencing protocl for MDA amplicon validation
  • This protocol also includes the S1 nucelase debranching and DNA Pol.I repairing treatments. I will compare the difference between with and without these treatments.

Plan

  • Prepare 2ug of E.coli gDNA MDA amplicon for each reaction in 100uL volume
  • S1 digestion(20units) for 0min(no S1 enzyme), 30min, 60min
  • Aliquote 80uL of digested DNA for sonification into 1~2 kB (Keep another 20uL for comparison)
  • Agarose gel size select the 1~2kB region. Recover DNA using Zymo kits or Montage filter.

Procedures

S1 nuclease digestion (debranching)

                     1rxn      6.5rxn
 DNA(666.ng/uL)       3.0      19.5
 S1(10U/uL, USB)      2.0       -
 2X S1 rxn buffer    50.0     325.0
 H2O                 45.0     292.5
 ----------------------------------
                    100.0     637.0 / 6.5 = 98 --- add S1 2uL or H2O 2uL
  • Incubate at PCR thermocycler for 30min(2rxn) and 60min(2rxn).
  • When incubation is completed, inactivate the reaction by adding 1uL 0.5M EDTA, vortex mixing, and incubating at 85C for 3min.
  • Store the S1-treated DNA in -20C.

Sonification

Sonification program:

Pulse on(30sec), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(2min).
  • Store the sheared DNA in -20C

Agarose gel size selection (with Zymo Gel DNA recovery kit)(09-26-'10)

Original gel
File:Sam092710-precut of sheared Ecoli amplicons.jpg

Post gel-cutting
File:Sam092710-postcut of sheared Ecoli amplicons.jpg
  • The cut gel slice (~100uL)was placed in 1.5-mL tube. Add 300uL ADB solution into tubes to dissolve the agarose gel.
  • Incubate at 55C in hotplate for 10min (with 500 rpm agitation). Briefly vortexing to mix the dissolved gel.
  • Transfer the melted agarose mixture onto Zymo-spin column in a collection tube. Centrifuge at 10,000xg for 30sec. Discard the flow-through.
  • Add 200uL wash buffer (added EtOH into wash buffer first). Centrifuge at 10,000xg for 30sec. Discard the flow-through.
  • Repeat the washing step again.
  • Add 15uL pre-warmed clean water on the filter. Incubate at RT for 1min. Transfer the filter on a clean 1.5-mL tubes. Centrifuge at 10,000xg for 1min to elute the DNA.

Blunt reactions (QUICK blunting kit, NEB)(09-26-'10)

  • Samples arrangemetns:
                     No.1  No.2  No.3  No.4  No.5  No.6
-------------------------------------------------------
S1 treatment         0m    0m    30m   30m   60m   60m
Pol.I treatment(1uL) -     yes   -     yes   -     yes
  • Set up blunting reactions on the cold block:
              1rxn     6.5rxn
-----------------------------
H2O           4.0      26.00
10X buf       2.5      16.25
1mM dNTP      2.5      16.25
Blunt EZ(NEB) 1.0       6.50
Sample       15.0       -
-----------------------------
             25.0      65.0/6.5 =10 --- Add DNA sample 15uL
  • Briefly vortexing the blunting reaction: Incubate at RT for 30min
  • Directly purify the Blunt reaction products using QIAquick without inactivation step.
    • Mix 5Vol. PB buffer(125uL) with Blunted products(25uL) and then follow the purification procedures.
    • Elute the purified DNA using 15uL prewarmed H2O.
Nanodrop results:
File:Sam092610 nanodrop.jpg

QIAquick purification and Blunt ligation(09-27-’10)

  • I am not sure if the blunted products could be used directly for ligation, so I purified both blunted reaction using QIAquick.
  • In order to concentrate my samples, I pool the duplicate sample into the same tube before purification (ligation no1 + no2 => 50uL, ligation no.3 + no.4 => 50uL)
  • Add another 50uL to make 100uL. Use 100uL as base volume for QIAquick purification.
  • DNA were inlute in 20uL H2O.
  • Nanodrop the concentration:
  No.1   No.2   No.3   No.4   No.5   No.6
  9.66   5.89   9.67   8.27   9.22   8.90 - ng/uL
  ------------------------------------------------
  1.55   2.54   1.55   1.81   1.62   1.68 - Amount need for 15ng Insert
  5.45   4.46   5.45   5.19   5.38   5.32 - Amount of H2O needed to make up to 7uL
  
                        No.1   No.2   No.3   No.4   No.5   No.6
---------------------------------------------------------------
Blunted-product(15ng)   1.55   2.54   1.55   1.81   1.62   1.68 
H2O (fill up to 7uL)    5.45   4.46   5.45   5.19   5.38   5.32             

                        1rxn    6.5rxn
10X Ligation buf.       1.00    6.50      
pCR-Blunt (25ng/uL)     1.00    6.50      
T4 DNA-ligase(4U/uL)    1.00    6.50      
------------------------------------------------
                        3.00   19.50/6.5=3uL --- Transfer to each of reaction tubes that contained 7uL samples.
  • Incubate at 16C for 1hr using PCR thermocycler. Perform transfection using the ligation product as soon as possible.

Heat-shock transfection

  • Equilibrate water bath to 42C.
  • Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
  • Thaw the TOP10 E.coli competent cell vial on ice.
  • Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocol).
  • Set up conpetent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins.
  • Adjust the timer: set up 30sec and 2min.
  • Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
  • Add 250ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall. No need for pipetting).
  • Shake the vials horizontally at 37C for 1 hour using vortexor in the incubator at the lowest speed.
  • Set up LB agar plates during the 1hr incubation.
  • After 1hr shacking mixing, plate 50uL from each transformation vial on agar plates (w/ Kanamycine).
    • There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design.
  • Incubate plates at 37C for about 20hr.
  • NOTE: I plated 12 plates in total. Two plates per sample. Incubation started from 2:00PM 09-27-'10.
Results (checked on 09-28-'10 12:00PM)

Sample    colonies count(white)  NOTE
-----------------------------------------
No.1-1       20    S1(0m)  - No DNA Pol.I
No.1-2       26    S1(0m)  - No DNA Pol.I
No.2-1       22    S1(0m)  - W/ DNA Pol.I
No.2-2       43    S1(0m)  - W/ DNA Pol.I   
No.3-1       45    S1(30m) - No DNA Pol.I
No.3-2       30    S1(30m) - No DNA Pol.I
No.4-1       10    S1(30m) - W/ DNA Pol.I
No.4-2       16    S1(30m) - W/ DNA Pol.I
No.5-1       44    S1(60m) - No DNA Pol.I
No.5-2       38    S1(60m) - No DNA Pol.I
No.6-1       66    S1(60m) - W/ DNA Pol.I
No.6-2       34    S1(60m) - W/ DNA Pol.I
-----------------------------------------
  • In general, the colonies number are fewer than I expected. It is possible that larger size insert is more difficult to clone. I will need to optimize the insert/vector ratio in the future.
    • The current ligation protocol works great for short inserts (200~400bp). For long insert (1kB) I may need to decrease amount to half for the reaction. I am curious if using only 8ng inserts can improve the results.
  • Except sample No.4, it looks like with S1 digestion treatment the successful cloning rate is a little bit increased.
    • The S1 digestion removes the single strand DNA, which leaves dsDNA for more blunting ligating.

Cloning PCR using M13 primer (provided in Zero Blunt PCR cloning kit)(09-28-'10)

  • The reason using M13 primer is due to confirm the existence of insert before sequencing. Besides, sequencing PCR products is cheaper than directed sequencing bacteria plasmid in Genewiz.
    • M13 primer binding sites are flanking on the insert region. Additional margin size ~200bp.
    • Since the M13 primer provided with kit are in 20uM, I will mix M13-f and M13-r together at 1:1 ratio to get the 10uM (f+r) mixture.
  • Prepare 10uL H2O in PCR tube. Spike the colonies into H2O and stir a little bit.
  • Incubate at 94C for 10min in thermocycler
  • I picked up four colonies (only from the 1st duplicate plates) from each of different conditions (no.1~no.6). In total 4x6=24 colonies were picked for clonyl PCR reactions.
Taq-DNA polymerase PCR recipe

                         1rxn   (8+1)x3=27rxn
--------------------------------------
Template                 2       -    ------ heat denatured colonies
M13 Primers (10uM, f+r)  1       27  
H20                     22      594
Taq 2x Maxter Mix       25      675
--------------------------------------
                        50     1296/27=48 ---- 2uL Template

Running at Program “PCR60” (Tm=60, 30cycle)
94C 3min -> (94C 30sec -> 60C 30sec -> 72C 1min) x 30cycle -> 72C 5min -> 15C forever
  • Basically I use the standard PCR protocol with a longer extension time(1min)
  • 10uL PCR products (leave 40uL for PCR product sequencing)were evaluated using 2% agasrose E-gel(run 12min)
Results: 

File:Sam092810-PCR validation no1-no4.jpg File:Sam092810-PCR validation no5-no6.jpg