Crm at 19:15, 8 October 2018

2018-10-08T19:15:31Z

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== Recipes ==

'''5X HF Phusion Buffer 1  '''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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'''  

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| Name
| Source 
| Stock 
| 500 ml
| 100 ul
| Final 
|-
| Tris<s>-HCl</s> 
| 
| 1.5 M
| 
| 
| 100 mM , pH <s>8.8 </s>, 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

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| Name
| Source 
| Stock 
| 500 ml
| 100 ul
| Final 
|-
| Tris<s>-HCl</s> 
| 
| 1.5 M
| 
| 
| 100 mM , pH <s>8.8 </s>, 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 

[[File:Kfu Buffer Screening.JPG|RTENOTITLE]]

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

[[File:Phusion BUffer Screening.JPG|RTENOTITLE]]

Old Kfu stock

[[File:Kfu Buffer Screening2.JPG|RTENOTITLE]]

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

[[File:Kfu KOD Buffer Screening.JPG|RTENOTITLE]]

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:

[[File:Taq Kfu Buffer screening.JPG|RTENOTITLE]]

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

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

Q5 Kfu Phusion Pfu Polymerase Buffer - Revision history

Crm at 19:15, 8 October 2018