User:David Benjamin Nyer/Notebook/PcTF breast cancer/2016/06/22: Difference between revisions

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==Summary==
==Summary==


Summarize what happened today. Put methods, data, and next steps in sections below.
Received four polymers from Kaushal lab for transfecting stubborn cell lines. Going to try them out at varying ratios of polymer:DNA with the MCF10A cell lines.
 
==Polymers==
 
Four polymers received:
* AGC18 (1:5): apramycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
* NGC18 (1:5): neomycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
* NR: neomycin monomer with resorcinol diglycidyl ether cross-linkers.
* PGC18 (1:5): paromomycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
 
==Procedure==
 
All polymers were received as 30 mg aliquots, solid, in glass test tubes. Samples are stable at -20 C for at least 6 months in this form.
 
<br><b>Prepare polymer stock solutions</b><br>
Need to make a 2 mg/mL stock solution for each polymer. Do so by weighing out 2 mg in a tared 1.5 mL centrifuge tube, then adding 1 mL of 1x PBS. Polymer NR is slightly hydrophobic, may require resuspension by incubation at room temperature on a rotator for 20-30 minutes.
 
<br><b>Prepare DNA:polymer conjugates</b><br>
Try using 10:1, 25:1 and 50:1 ratios of polymer:DNA for the screen. See the chart below:
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample ID'''
| align="center" style="background:#f0f0f0;"|'''Polymer type'''
| align="center" style="background:#f0f0f0;"|'''Ratio'''
| align="center" style="background:#f0f0f0;"|'''Polymer amount (µL)'''
| align="center" style="background:#f0f0f0;"|'''DNA type'''
| align="center" style="background:#f0f0f0;"|'''DNA amount (µg)'''
| align="center" style="background:#f0f0f0;"|'''DNA amount (µL)'''
| align="center" style="background:#f0f0f0;"|'''PBS amount (µL)'''
|-
| 1||AGC18 (1:5)||1:10||10||KAH126-MV2||2||4||86
|-
| 2||AGC18 (1:5)||1:25||25||KAH126-MV2||2||4||71
|-
| 3||AGC18 (1:5)||1:50||50||KAH126-MV2||2||4||46
|-
| 4||NGC18 (1:5)||1:10||10||KAH126-MV2||2||4||86
|-
| 5||NGC18 (1:5)||1:25||25||KAH126-MV2||2||4||71
|-
| 6||NGC18 (1:5)||1:50||50||KAH126-MV2||2||4||46
|-
| 7||NR||1:10||10||KAH126-MV2||2||4||86
|-
| 8||NR||1:25||25||KAH126-MV2||2||4||71
|-
| 9||NR||1:50||50||KAH126-MV2||2||4||46
|-
| 10||PGC18 (1:5)||1:10||10||KAH126-MV2||2||4||86
|-
| 11||PGC18 (1:5)||1:25||25||KAH126-MV2||2||4||71
|-
| 12||PGC18 (1:5)||1:50||50||KAH126-MV2||2||4||46
|}
 
Combine DNA and polymer in each of 12 labeled tubes, then allow to incubate at room temperature for 30 minutes.
 
<br><b>Transfect Cells</b><br>
Add polymer:DNA conjugate to respective wells of cells in a 6-well plate. Mix by rocking the plate, then centrifuge at 100 g for 5 minutes. Return to CO2 incubator.




Revision as of 16:21, 22 June 2016

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Summary

Received four polymers from Kaushal lab for transfecting stubborn cell lines. Going to try them out at varying ratios of polymer:DNA with the MCF10A cell lines.

Polymers

Four polymers received:

  • AGC18 (1:5): apramycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
  • NGC18 (1:5): neomycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
  • NR: neomycin monomer with resorcinol diglycidyl ether cross-linkers.
  • PGC18 (1:5): paromomycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.

Procedure

All polymers were received as 30 mg aliquots, solid, in glass test tubes. Samples are stable at -20 C for at least 6 months in this form.


Prepare polymer stock solutions
Need to make a 2 mg/mL stock solution for each polymer. Do so by weighing out 2 mg in a tared 1.5 mL centrifuge tube, then adding 1 mL of 1x PBS. Polymer NR is slightly hydrophobic, may require resuspension by incubation at room temperature on a rotator for 20-30 minutes.


Prepare DNA:polymer conjugates
Try using 10:1, 25:1 and 50:1 ratios of polymer:DNA for the screen. See the chart below:

Sample ID Polymer type Ratio Polymer amount (µL) DNA type DNA amount (µg) DNA amount (µL) PBS amount (µL)
1 AGC18 (1:5) 1:10 10 KAH126-MV2 2 4 86
2 AGC18 (1:5) 1:25 25 KAH126-MV2 2 4 71
3 AGC18 (1:5) 1:50 50 KAH126-MV2 2 4 46
4 NGC18 (1:5) 1:10 10 KAH126-MV2 2 4 86
5 NGC18 (1:5) 1:25 25 KAH126-MV2 2 4 71
6 NGC18 (1:5) 1:50 50 KAH126-MV2 2 4 46
7 NR 1:10 10 KAH126-MV2 2 4 86
8 NR 1:25 25 KAH126-MV2 2 4 71
9 NR 1:50 50 KAH126-MV2 2 4 46
10 PGC18 (1:5) 1:10 10 KAH126-MV2 2 4 86
11 PGC18 (1:5) 1:25 25 KAH126-MV2 2 4 71
12 PGC18 (1:5) 1:50 50 KAH126-MV2 2 4 46

Combine DNA and polymer in each of 12 labeled tubes, then allow to incubate at room temperature for 30 minutes.


Transfect Cells
Add polymer:DNA conjugate to respective wells of cells in a 6-well plate. Mix by rocking the plate, then centrifuge at 100 g for 5 minutes. Return to CO2 incubator.



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