Daniel:Notebook/HiResChrPaint/2013-9-26: Difference between revisions

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>Djacobse
(Created page with "=Amino Group Primers= Back to Calendar So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group,...")
 
>Djacobse
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  2. Aliquot 100 uL into each of 5 qPCR tubes (20 in total)
  2. Aliquot 100 uL into each of 2 qPCR tubes (8 in total)
  3. PCR protocol:
  3. PCR protocol:
     i. PCR 3 min 95C
     i. PCR 3 min 95C
Line 96: Line 96:
     vii. 72C 2 min
     vii. 72C 2 min
     viii. 16C hold
     viii. 16C hold
  4. Pool 3 samples together and 2 samples together (300, 200 uL each)
  4. Pool 2 samples together (200 uL each)
5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB
6. Measure in nanodrop
 
===Dye Coupling===
 
*Following [[Daniel:Protocols/AlexaDyeCouple|ARES Protocol]]
 
0. Dry samples in vacuum centrifuge
1.

Revision as of 22:56, 26 September 2013

Amino Group Primers

Back to Calendar

So the new primers are in. We ordered them a few weeks ago. All the primers have a 5' amino group, which should be compatible with the Alexa-dye coupling system. Thanks to Matt's results yesterday, I know that when the dye coupling does not work, the Centri-Sep columns yield very little dye. So, before going nuts on making new probes, I'm going to test the water with a simple comparison. For sample 1, I'm going to test the dye using the new primer, plus no aa-dUTP. I'm also going to do a positive control using a standard amplification using the old primers and aa-dUTP. Finally, I'll run a negative control using old primer and no aa-dUTP. I'll do this for both V4S3 and V6S3, since I should test both primer sets.

Amino Primers Test

qPCR

1. Reaction table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U 100 uM AP2V4 100 uM AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
std 100 uL rxn 0.2 50 0.4 0.4 0.4 0.4 2.5 46 100
V4S3* (2.5x) 0.5 125 1 1 0 0 0 122.5 250
V6S3* (2.5x) 0.5 125 0 0 1 1 0 122.5 250
V6S3 (2.5x) 0.5 125 0 0 1 1 6.25 116.25 250
V6S3- (2.5x) 0.5 125 0 0 1 1 0 122.5 250
2. Aliquot 100 uL into each of 2 qPCR tubes (8 in total)
3. PCR protocol:
    i. PCR 3 min 95C
    ii. 95C 3 sec
    iii. 55C 30 sec 
    iv. 72C 20 sec
    v. plate read
    vi. goto ii
    vii. 72C 2 min
    viii. 16C hold
4. Pool 2 samples together (200 uL each)
5. Column Purification, elute with 40 uL EB
6. Measure in nanodrop

Dye Coupling

0. Dry samples in vacuum centrifuge
1.