Editing Plasmid buffers (section)

== <br/><u>'''Mini-prep'''</u> ==

<br/><br/>'''Buffer P1'''<br/><br/>50 mM Tris HCl, pH 8.0, 10 mM EDTA, (or 2. 5 ml of 100X Tris-EDTA buffer from Sigma #T-9285 in 50 ml water)<br/>100 ug/ml RNase A.

'''P1''' from another protocol: 10 mM Tris-HCl, pH8, 100 ug/ml RNase A.

6.06 g Tris Base, 3.72 g Na<sub>2</sub>EDTA.2H<sub>2</sub>O in 800 ml H2O, adjust the pH with HCl, add water to 1 L, add 100 mg RNase A per liter of P1.

add some lyseblue (Thymophthalein)





'''Resuspension Buffer''': 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 ug/mL RNase A<br/>'''Lysis Buffer''': 200 mM NaOH, 1% SDS<br/>'''Precipitation Buffer''': 4.2 M guanidine hydrochloride, 0.9M Potassium acetate pH 4.8<br/>'''Equilibration Buffer''': 7M Gu-HCl pH 5.5<br/>'''Wash 1 Buffer''': 5M guanidine HCl, 30% Ethanol, 10 mM TRIS-HCl pH 6.6<br/>'''Wash 2 Buffer''': 10 mM TRIS-HCl pH 7.5, 80% Ethanol<br/>'''Elution Solution''': Water<br/>'''RNase A''': Dissolve RNASE A powder in H2O to give 100 mg/ml. Boil 5 min to kill DNase. Aliquot and store at -20 oC.<br/><br/><br/>'''Buffer P2''' (200mM NaOH, 1% SDS):<br/>1M NaOH 10 ml<br/>10% SDS 5 ml<br/>Water 35 ml

Dissolve 8.0 g NaOH in 950 ml dH2O, 50 ml 20% SDS (w/v) solution.&nbsp; The final volume should be 1 liter.<br/>Some protocols use 2% SDS.

'''Buffer P3'''

Dissolve 294.5 g potassium acetate in 500 ml dH2O.&nbsp; Adjust the pH to 5.5 with glacial acetic acid (~110 ml).&nbsp; Adjust the volume to 1 liter with dH2O.

3.0 M potassium acetate pH 5.5<br/>can be stored either at room temp or 2~8°C



'''PB'''

5 M guanidine hydrochloride (MW 95.53 ), 20 mM Tris-HCl, pH 6.6, (25 oC), add ethanol to 38%.

200 ml:

95.53 g guanidine hydrochloride

8 ml of 0.5M Tris-HCl, pH6.6

76 ml of ethanol



'''PB from another protocol:''' 5 M Gu-HCl, 30% <strike>isopropanol</strike> ethanol

<br/>'''Buffer DP3''' (for Qiagen Directprep 96-well miniprep)

3.0 M ammonium acetate pH 5.5



'''PB Buffer''': 5 M guanidine hydrochloride, 30%<strike>isopropanol</strike>.ethanol



'''Buffer FWB'''

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH2O.&nbsp; Adjust the pH to 7.0 with NaOH.&nbsp; Add 150 ml pure isopropanol and adjust the volume to 1 liter with dH2O.



'''Buffer QBT'''

Dissolve 43.83 g NaCl, <strike>10.46 g MOPS (free acid) .</strike>&nbsp; 7g Tris-HCl, 0.5 g Tris Base in 800 ml dH2O. <strike>Adjust the pH to 7.0 with NaOH.&nbsp;</strike> Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with dH2O.

750 mM NaCl<br/>50 mM <strike>MOPS </strike>Tris-HCl, pH 7.0<br/>15% isopropanol (v/v)<br/>0.15% Triton® X-100 (v/v)



'''N3-Ca''': N3 with 1.4 M CaCl<sub>2</sub>&nbsp;for precipitate high molecular weight&nbsp;RNA

'''2X PEG6000-NaCl<sub>2</sub>'''&nbsp;, 20% PEG 6000, 1.6 M NaCl.&nbsp;&nbsp;for precipiate plasmid DNA: 4 ml of 50&nbsp;% PEG 6000, 4 ml of 4 M NaCl, 2 ml H<sub>2</sub>O, autoclave.



'''Buffer QC'''

Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O.&nbsp; Adjust the pH to 7.0 with NaOH.&nbsp; Add 150 ml pure isopropanol.&nbsp; Adjust the volume to 1 liter with dH2O.

1.0 M NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol (v/v)



'''Buffer QF'''

'''From Qiagen website'''

Dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml dH2O and adjust the pH to 8.5 with HCl.&nbsp; Add 150 ml pure isopropanol.&nbsp; Adjust the volume to 1 liter with dH2O.

1.25 M NaCl<br/>50 mM Tris·Cl pH 8.5<br/>15% isopropanol (v/v)



Store at 15–25°C


73.05 g&nbsp;NaCl

2.21<span style="white-space:pre">g </span>Trizma HCL

4.36 g&nbsp;Trizma Base



'''Buffer QN'''

'''from Qiagen website'''

Dissolve 93.50 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O and adjust the pH to 7.0 with NaOH.&nbsp; Add 150 ml pure isopropanol.&nbsp; Adjust the volume to 1 liter with dH2O.

1.6M NaCl<br/>50 mM MOPS pH 7.0<br/>15% isopropanol





'''Buffer STE'''

Dissolve 5.84 g NaCl, 1.21 g Tris base, and 0.37 g Na2EDTA*2H2O in 800 ml dH2O.&nbsp; Adjust the pH to 8.0 with HCl.&nbsp; Adjust the volume to 1 liter with dH2O.

<br/>'''Buffer P3'''<br/>3.0 M KOAc, pH 5.5 (29.5 g/ 100ml water, adjust pH with glacial acetic acid )





'''N3 from another protocol''':

4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to pH4.2~4.8 by adding acetic acid.

200 ml:

80 g guanidine hydrochloride

18g potassium acetate

10 ml acetic acid (add this inside the hood),

add DI water to dissolve

add water to 200 ml.



Yet another protocol

<br/>4M guanidine hydrochloride (GuHCl)<br/>0.5M potassium acetate<br/>pH 4.2



'''Improved N3''' 4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to pH4.2~4.8 by adding acetic acid.

200 ml:

80 g guanidine hydrochloride

18g potassium acetate

10 ml acetic acid (add this inside the hood),

'''6 ml of 1 M MgCl2'''

'''CaCl<sub>2</sub> 1.4 M'''



add DI water to dissolve

add water to 200 ml.

Buffer N3

4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8 Buffer PB

5 M Gu-HCl 30% isopropanol Buffer QG

5.5 M guanidine thiocyanate (GuSCN) 20 mM Tris HCl pH 6.6 Buffer PE

10 mM Tris-HCl pH 7.5 80% ethanol

'''Buffer QXB''' (for binding of large >3000 bp fragments to columns)

5 M GuHCl

<br/>'''Buffer PE'''<br/>80% EtOH.<br/>25 mM NaCl,<br/>5 mM Tris-HCl, pH 7.5

5 ml autoclaved Tris-HCl, pH 8.0

6 ml 4 M autoclaved NaCl

Autoclaved dd H<sub>2</sub>O to 250 ml

Add 800 ml EtOH<br/><br/>'''Buffer EB'''<br/>10 mM Tris·Cl, pH 8-8.5<br/>make up to 1 liter with dH20