Brandon:Protocols/Lysis buffers: Difference between revisions

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Line 8: Line 8:
  100mM NaCl
  100mM NaCl
  30mM MgCl2
  30mM MgCl2
  1% NP40
  1% IGEPAL® CA-630 (replaces NP40)


*make 1M solution of NaCl and MgCl2
*make 1M solution of NaCl and MgCl2
  1M NaCl2, 2.922 grams in 50 mL nuclease free water
  1M NaCl, 1.461 grams in nuclease free/DI water and bring to 25 mL
  1M MgCl2, 4.761 grams in 50 mL nuclease free water (add mgcl2 to water, very exothermic!!)
  1M MgCl2, 2.3805 grams in nuclease free/DI water and bring to 25 mL (add mgcl2 to water, very exothermic!!)


*To make 10 mL of lysis buffer
*To make 10 mL of lysis buffer
Line 18: Line 18:
  1000  uL of 1M NaCl
  1000  uL of 1M NaCl
  333  uL of 1M MgCl2
  333  uL of 1M MgCl2
  10   uL of NP40 (1% in soln, NP-40 substitute is fine)
  100   uL of IGEPAL® CA-630 (1% in soln, replaces NP40)
  8.067 mL Nuclease free water
  8.067 mL Nuclease free water
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''10X'''
| align="center" style="background:#f0f0f0;"|'''100X'''
| align="center" style="background:#f0f0f0;"|'''50X'''
| align="center" style="background:#f0f0f0;"|'''40X'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| Tris-HCl pH 7.5||2.0M||500||5000||2500||2000||uL
|-
| NaCl||5M||200||2000||1000||800||uL
|-
| MgCl||1M||333||3330||1665||1332||uL
|-
| IGEPAL CA-630||solid||100||1000||500||400||uL
|-
| ||||||0||0||0||uL
|-
| ||||||||||||
|-
| ||||||||||||
|-
| ||||||11330||5665||4532||uL
|-
| ||||H20||-1330||4335||5468||uL
|-
|
|}

Latest revision as of 20:03, 21 November 2014

lysis buffers[edit]

lysis buffer 1[edit]

  • Crawford et al. PNAS 2003
  • lysis buffer composition:
10X lysis buffer 
100mM Tris.Hcl pH 7.5
100mM NaCl
30mM MgCl2
1% IGEPAL® CA-630 (replaces NP40)
  • make 1M solution of NaCl and MgCl2
1M NaCl, 1.461 grams in nuclease free/DI water and bring to 25 mL
1M MgCl2, 2.3805 grams in nuclease free/DI water and bring to 25 mL (add mgcl2 to water, very exothermic!!)
  • To make 10 mL of lysis buffer
500   uL of 2M Tris.Hcl PH7.5
1000  uL of 1M NaCl
333   uL of 1M MgCl2
100   uL of IGEPAL® CA-630 (1% in soln, replaces NP40)
8.067 mL Nuclease free water
' ' 10X 100X 50X 40X '
Tris-HCl pH 7.5 2.0M 500 5000 2500 2000 uL
NaCl 5M 200 2000 1000 800 uL
MgCl 1M 333 3330 1665 1332 uL
IGEPAL CA-630 solid 100 1000 500 400 uL
0 0 0 uL
11330 5665 4532 uL
H20 -1330 4335 5468 uL