Dinh:Protocols/Probes Prep Nov24

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Revision as of 19:11, 11 December 2009 by >Sam Chiang (→‎Step 4: QiaQuick Purification)
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November 24, 2009

KAPA SYBR FAST qPCR Kit Master Mix (2x) Universal

ideally suited for gene expression analysis
low copy gene detection
microarray validation
gene knockdown validation
novel enzyme in kit enhances the amplification efficiency of both high
 GC and AT templates

Notes:

protect kit components from light
always ensure that the product has been fully thawed and mixed before use
do not exceed 25 ul reaction volumes
do not exceed 20ng template DNA per 24 ul reaction volume
Chromo4 RT Detector does not require ROX Reference dye

Objectives:

To use KAPA qPCR kit on CpG30K probes set, using reaction volume of
 25ul and 100ul
To practice QUBIT DNA quantification
To create a probes preparation/purification table

Previous methods to quantify probes concentration at each purification steps included Nanodrop and PAGE quantification. However, Nanodrop quantification is inaccurate after ethanol precipitation. The drawback to PAGE quantification is that it cannot determine the total DNA concentration. I will use QUBIT and PAGE to determine the efficiency of each probes preparation step.

November 25, 2009

Step 1: PCR Amplification

Reaction Mix (100ul reaction volume):

Reagent Final Conc. Vol (1x) Vol (8x) Vol (100x)
ddH20 49.4ul 395ul 4940ul
2x KAPA qPCR Master Mix 1x 50ul 400ul 5000ul
AP1V4IU (100uM) 0.3uM 0.3ul 2.4ul 30ul
AP2V4 (100uM) 0.3uM 0.3ul 2.4ul 30ul
Template CpG30k (20nM) 33pg 0.05ul 0.4ul 5ul
Total 100.05ul 800.4ul 10005ul

Program

94C - 30 s
94C - 3 s
60C - 30 s
Plate Read
Cycle to 2 for 29 more times
72C - 5 min
15C - forever
end

Protocol:

Place KAPA 2x qPCR MM on ice.
Add ddH20, primers, and template to 15ml tube (RNAase/DNAase free). Vortex & leave on ice for 2-3 min
Prepare strip tubes on cooling rack
Add KAPA 2x MM & vortex, shake down reagents, then pour into 55ml solution basin
Use multichannel p-100 pipettor. Pipette 101ul to each tube. For last row, use p-200 to pipette 101ul to each tube.
Place caps on strip tubes
Spin pairs of strip tubes for 5-10 s to remove bubbles
Prepare new run on Chromo4 RT-PCR machine, let it get to 94 C
Pause program, place tubes in machine, and resume run

3. Stopped at 19 cycles.

Notes:

8x reaction mix was prepared as test run. 100ul was aliquoted to 6 tubes and amplified using RT-PCR for 26 cycles (over-amplified), amplification slope started at cycle 13 and plateaued at cycle 25. For the rest of the reaction mix, 25ul was aliquoted to 8 tubes. Only 4 tubes were amplified using the the same program for 26 cycles (over-amplified), amplification sloped started also at cycle 13 and plateaued at cycle 25. Concluded no difference between 25ul and 100ul reaction volumes.

The leftover 4 tubes of 25ul reaction volume was used in another run:

Program

95C - 30 s
95C - 3 s
60C - 30 s
72C - 3 s
Plate Read
Cycle to 2 for 29 more times
72C - 5 min
15C - forever
end
Recommended by KAPA qPCR manual: 95C denaturation (initital & in cycle), and 72C during plate read. 

However, for this run, amplification slope started at cycle 15 and plateaued also at cycle 23. Concluded that other program used was better suited for this probe set.

Step 2: Ethanol Precipitation

Performed as in previous protocols using 8x15ml tubes.

Step 3: Lambda Exonuclease

All dissolved products in RNase free dH20 was pooled.

130ul sample, 15ul 10x lambda exonuclease buffer, 5ul lambda exonuclease. 
8 reactions.

Step 4: QiaQuick Purification

Performed as in previous protocols. All reactions were first pooled and 150 ul was aliquoted to 7 tubes containing 750ul buffer PE. Divided solution equally to two columns.

Tested EB vs DNase Free H20

~130ul was aliquoted to 1 tube containing 750ul buffer PBI. 
Divided solution equally to two columns. 
1 column was eluted with elution buffer (EB). 
1 column was eluted with DNase free dH20.

Step 5: USER Digestion

Performed as in previous protocols. Digested for 3 hours.

3ul USER Enzyme
7ul 10x DpnII buffer

Step 6: DpnII Digestion

3ul 100nM DpnII oligo 
1ul 10x DpnII buffer

Step 7: PAGE Size selection

Performed as in previous protocols. Pooled all reactions. 100ul sample + 100ul loading dye per 2D well, 6x2D gels.

Concentration of ssDNA in solution was 50.9ng/ul x 100ul loaded = 5090ng loaded per 2D well.

Step 8: EtOH ppt

Pooled all flowthrough and divide equally to 10x1.5mL tubes. Then during washing step, combing blue pellets to one tube

*Since all tubes are expected to have approx. the same DNA mass, if no blue pellet is seen:
add 0.5ul Glycoblue, vortex, and re-freeze solution at -70C for 20min.

Step 9: Quantification

Lane STEP Volume Loaded(ul) Qubit Conc. (ng/ul) Dilution Mass Loaded
1 After EtOH ppt 1 103 1 103
2 After Lambda Exonuclease 1 55.25 1 55.25
3 After QiaQuick 1 83.42 1 83.42
4a After USER/DpnII 0.6 50.9 1 30.54
4b After USER/DpnII 0.3 50.9 1 15.27
W After USER/DpnII(water diluted in last QQ step) 1 <50.9 1 -
E After USER/DpnII(EB diluted in last QQ step) 1 <50.9 1 -
  1. The use of EB does not negatively affect digestion or yield.







Purification Table

Step Volume (ul) Purity Conc. (ng/ul) ssDNA (ng) % Recovery
1 After first EtOH ppt 1020 9.41 103 105060
2 After lambda exon 1103 0.92 55.25 60941 58.01%
3 After qiaquick 420 2.63 83.42 35035 57.49%
4 After USER &DpnII 596 0.7 50.9 26522(*) 75.70%
5 After size selection & Nanosep 4047 0.13 0.33 1315 4.96%
6 After final EtOH ppt 20 202.350 23.48 470 35.70%
  1. Purification factor: previous step volume/current step volume
  2. % Recovery: previous step mass/current step mass
  3. (*)Subtracted mass of oligo added: 100nM*4238g/moles*1/1000*3ul/rxn*9rxns = 3800ng


Conclusions