Daniel:Notebook/ComboLock/2016-11-7: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
(One intermediate revision by the same user not shown)
Line 130: Line 130:
<li>Add 20 uL Phusion mix to 20 uL of reaction for sample 1</li>
<li>Add 20 uL Phusion mix to 20 uL of reaction for sample 1</li>
<li>Incubate at 55C for 2 hours</li>
<li>Incubate at 55C for 2 hours</li>
<li>Continued [[Daniel:Notebook/ComboLock/2016-10-22|tomorrow]]</li>
<li>Heat kill for 2 minutes at 94C and hold at 16C overnight; Continued [[Daniel:Notebook/ComboLock/2016-11-8|tomorrow]]</li>
</ol>
</ol>



Latest revision as of 18:36, 8 November 2016

Cell Test with RCA[edit]

Back to Calendar

This experiment is designed to see if I can get mRNA targeting C-Probes to work in cells, this time with RCA. I'll be trying both VIM and SOD1, just to have two separate genes.

Buffer Prep[edit]

Need more C Probe Hybridization Buffer. I'll be freezing it down in aliquots of 500 uL.

Reagent Stock Final Dilution Amount in 10 mL (uL)
SSC Buffer 20X 1X 20 500
Polyvinylsulonic Acid 100% 2.50% 40 250
Ribonucleoside Vanadyl Complex 200 mM 20 mM 10 1000
Rnasin 40000 U/mL 40 U/mL 1000 10
0.1% Tween-20 10% 0.10% 100 100
Salmon Sperm DNA 10 mg/mL 100 ug/mL 100 100
DEPC Treated Water NA NA NA 8040

Protocol[edit]

Sample Matrix

  C Probes-VIM /SOD1 (C1v2+C2) U87MG
Sample 1 (AB) X X
Sample 2 (AB) X X
Sample 3 (AB)   X
Sample 4 (AB) X  


  1. Cell Preparation
    1. Pellet cells via centrifugation at 600g for 3 min
    2. Wash with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    3. Add 100 uL 50% hematoxylin (filtered) and pellet cells via centrifugation at 600g for 3 min
    4. Wash once with 200 uL wash buffer and pellet cells via centrifugation at 600g for 3 min
    5. Wash 2 times with 200 uL wash buffer prime and pellet cells via centrifugation at 600g for 3 min
    6. Pellet cells via centrifugation at 600g for 3 min
    7. Combine 0.5 uL of each C probe into a 0.2 mL tube with 2 uL nfH2O
    8. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    9. Add probes to CPH buffer according to sample matrix, 1 uL probe mix per sample
    10. Incubate probes at 40C for 1 hour with vigorous agitation
    11. Wash three times with 200 uL wash buffer and pelleting at 600xg for 3 min
    12. Incubate cells for 20 min at 40C in 200 uL stringent wash buffer
  2. Latch and Padlock Hybridization
    1. Pellet at 600xg for 3 min and remove supernatant
    2. Resuspend pellet in 100 uL LPH buffer with 100 nM Padlock0201-68 and 100 nM Latch0001
    3. Incubate for 30 min at 37C
    4. Wash twice with 100 uL wash buffer by pelleting cells at 600xg for 3 min
    5. Incubate cells for 20 min at 40C in 150 uL stringent wash buffer
    6. Resuspend with 20 uL 1x Amp Ligase buffer and transfer to a 0.2 mL tube
  3. Circularization
    1. Prepare 8.2X uL Phusion mix
    2. Add 20 uL Phusion mix to 20 uL of reaction for sample 1
    3. Incubate at 55C for 2 hours
    4. Heat kill for 2 minutes at 94C and hold at 16C overnight; Continued tomorrow

    Buffers[edit]

    Phusion Mix

    Reagent Stock Conc Final Amount 1x Vol (uL) MM Vol (8.2x) (uL)
    NAD+ 5 mM 40 nmol 8 65.6
    dNTP 1 mM 600 pmol 0.6 4.92
    Betaine 5 M 15 umol 3 24.6
    10X AmpLigase Buffer 10X 1X 2 16.4
    Amp Ligase 5 U/uL 10 U 2 16.4
    Phusion HF DNA Polymerase 2000 U/mL 6.4U 3.2 26.24
    nf H2O     1.2 9.84
    Total     20 164

    ComboLock Wash Buffer

    Reagent Stock Final Dilution Amount in 20 mL
    PBS 10X 1X 10 2 mL
    SSC 20X 4X 5 4 mL
    RNAsin 40000 U/mL 4 U/mL 10000 2 uL
    nf H2O NA NA NA 14 mL