Q5 Kfu Phusion Pfu Polymerase Buffer

Recipes

5X HF Phusion Buffer 1  

Name	Source	Stock	500 ml	100 ul	Final
Tris		1.5M		10	150 mM , pH 10
KCl		1 M		5	50 mM
NH4OAc		1 M		5	50 mM
MgSO4		0.1 M		10	10 mM
Triton X-100		10%		5	0.5%
BSA		10mg/ml		5	0.5 mg/mL
H2O				60

 5X HF Kfu Buffer  2

Name	Source	Stock	500 ml	50 ml	100 ul	Final
Tris	Tris base, Sigma, ultrapure	1.5 M, pH 10.0, adjust to pH 10 with KOH	75 ml	7.5 ml	10	150 mM
K2SO4	Potassium sulfate, Sigma, ultrapure	0.5 M	200 ml	20 ml	40	200 mM
(NH4)2SO4	Ammonium sulfate, J. T. Baker ultrapure	0.1 M	25 ml	62.5 ul of 4 M	5	5 mM
MgSO4	Magnesium sulfate, Sigma, ultrapure	0.1 M	5 ml of 1 M	0.5 ml of 1 M	10	10 mM
Triton  X-100	Sigma, Ultrapure	10%	25 ml	2.5 ml 0f 10%	5	0.5%
H2O	Water, DNase- RNase-free, Ultrapure		ml	19.5 ml	30

5X   Kfu Buffer Recipe 3, the same as 2
 

Name	Source	Stock	500 ml	100 ul	Final
Tris		1.5M		10	150 mM , pH 10
K2SO4		0.5 M		40	200 mM
(NH4)2SO4		0.1 M		5	5 mM
MgSO4		0.1 M		10	10 mM
Triton X-100		10%		5	0.5%
H2O				30

5X   Kfu Buffer  4,  similar to 1,  with 1 M trehalose

Name	Source	Stock	500 ml	100 ul	Final
Tris					150 mM , pH 10
KCl					50 mM
NH4OAc					50 mM
MgSO4					10 mM
Triton X-100					0.5%
BSA					0.5 mg/mL
Trehalose					1 M
H2O

Buffer 5. (adapted from Buffer for Deep Vent), similar to buffer 1

Name	Source	Stock	500 ml	100 ul	Final
Tris-HCl		1.5 M			100 mM , pH 8.8 , 10
KCl		1 M			50 mM
(NH4)2SO4		0.1 M			50 mM
MgSO4		0.1 M			10 mM
Triton X-100		10%			0.5%
H2O

Buffer 6, Pfu buffer from Promega

Name	Source	Stock	500 ml	100 ul	Final
Tris-HCl		1.5 M			100 mM , pH 8.8 , 10
KCl		1 M			50 mM
(NH4)2SO4		0.1 M			50 mM
MgSO4		0.1 M			10 mM
Triton X-100		10%			0.5%
BSA		10 mg/ml			0.5 mg/ml
H2O

Experiement

PCR: 5 ul of each buffer, 1 ul of 10 mM dNTP, 1 ul of 10 uM 986-F1, 1 ul of 10 uM 986-R1, template 986, 1 ul Phusion or Kfu (12 ng/ul)

95oC for 2 min, 30 cycles of 94 oC 30, 55 oC 30, 72 oC 4 min, 72 oC 5 min.

Lanes are buffer, * with addtional 0.25 ul of 0.1 mg/ul Taq, #5 is 

Lanes are buffer, * with addtional 1 ul of 0.1 mg/ul Taq,  

Old Kfu stock

Newly prepared Kfu vs KOD, 12 ul or 24 ul of 1 mg/mg stock to 1 ml storage buffer 

20 mM HEPES (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, stabilizers (?), 200 µg/ml BSA and 50 % glycerol

Enzyme Prep: add 20 ul of 10 mg/ml BSA, 10 ul 1 M DTT, 12 ul of 1 mg/ml of Kfu to  storage buffer: 20 mM HEPES (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl,   and 50 % glycerol

10/8/2018

Screening buffer using Taq polymerase (0.1mg/ml) and Kfu. PCR template: 1708, oligos: 135-F and 135-R, size: 1200bp

Buffer 7 recipe:

Conclusion

Buffer 6 is good for Kfu, Buffer 2 is good for Phusion

5X HF Phusion Buffer Recipe 4

1000mM Trehalose
500mM Sarcosine
400mM KCl
80mM (NH4)2SO4
50mM Arginine
50mM POPSO
7.5mM MgCl2
0.5% Tween-20
5(-25%) Glycerol (depending on GC)
5(-15%) DMSO (depending on GC)
pH = 8.8

Additional components for Green Buffer (for direct Gel loading);
<12.5% (w/v) Ficoll 400 (as low as possible whilst still allowing for crisp bands)
0.03% (w/v) Tartrazine
0.003% (w/v) Xylene Cyanol

Novagen’s KOD buffer 1

from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC168769/
120 mM Tris-HCl (pH 8.0), 1.2 mM MgCl2, 6 mM (NH4)2SO4, 10 mM KCl, 0.1% Triton X-100, and 0.01% BSA for KOD DNA polymerase

https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-10-21

http://pipettejockey.com/2017/08/18/purifying-commonly-used-enzymes-homebrew-phusion/

Q5 5X PCR enhancer: 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA ,  stable at 20 C for at least 3 months, http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.553.4058&rep=rep1&type=pdf

Taq Buffer

 65 mM Tris–Cl, 16.6 mM (NH4)2SO4, 3.1 mM MgCl2, and 0.01% (v/v) Tween 20 at a pH of 8.8 a