Daniel:Notebook/HiResChrPaint/2013-10-4

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V6S3 and V4S1 Amplification Amino Primers (Started 10/03/2013)[edit]

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DpnII Digestion[edit]

3. Column purification, elute with 30 uL EB
4. Measure Concentration in nanodrop
5. Check digestion results with gel

Gel Results[edit]

File:20131004 DpnIIDigest.jpg

Nanodrop Results[edit]

  V4S1 V6S3AB V6S3CD
ng/uL 39.9 175.6 152
ug in 30 uL 1.2 5.3 4.6
ug total 1.2   9.8

Discussion[edit]

The V4S1 really doesn't have a lot of DNA left, so I'm going to stop here and amplify the V4 set again before I continue on.

V4S1 Amplification[edit]

qPCR[edit]

1. Reaction Table
  20 nM OligoPool 2X Kapa SYBR qPCR MM 100 uM AP1V41U-Amino 100 uM AP2V4 H20 Total Volume
std 100 uL rxn 0.2 50 0.4 0.4 49 100
V4S1 (12.5x) 2.5 125 5 5 612.5 1250
2. Aliquot 100 uL into each of 12 qPCR tubes (24 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 3 samples together (300 uL each)
5. Column Purification, elute with 40 uL EB
6. Measure in nanodrop

qPCR Results[edit]

File:2013-10-04-V4S1.png

Nanodrop Results[edit]

  V4S1A V4S1B V4S1C V4S1D
ng/uL 76.5 76.3 76.3 68.3
ug in 40 uL 3.1 3.1 3.1 2.7
ug total       11.9

DpnII Digestion[edit]

1. Reaction table
Sample V4S1A V4S1B V4S1C V4S1D
ng/uL 76.5 76.3 76.3 68.3
ug in 35 uL 2.7 2.7 2.7 2.4
uL 10X buffer 5 5 5 5
uL DpnII (50U/uL) 2 2 2 2
uL nfH2O 8 8 8 8
Total 50 50 50 50
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued tomorrow

CERC[edit]

Split cells today, including making two dishes for FISH.

FISH[edit]

From the dishes above.

Protocol[edit]

1. Rinse slides briefly in 1X PBS
2. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
3. Rinse briefly with 1 mL 1X PBS
4. Incubate for 5 minutes in 1 mL 2X SSCT
5. Incubate for 5 minutes in 1 mL 2X SSCT + 50% (v/v) formamide
6. Aspirate, and add 1mL of 2X SSCT + 50% (v/v) formamide for storage at 4C
7. Slides are typically best used within 1-2 weeks of creation