Editing Q5 Kfu Phusion Pfu Polymerase Buffer

== Recipes ==

'''5X HF Phusion Buffer 1&nbsp;&nbsp;'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Name
| Source<br/>
| Stock<br/>
| 500 ml
| 100 ul
| Final<br/>
|-
| Tris<br/>
| <br/>
| 1.5M
| <br/>
| 10&nbsp;
| 150 mM , pH 10<br/>
|-
| KCl<br/>
| <br/>
| 1 M
| <br/>
| 5
| 50 mM&nbsp;<br/>
|-
| NH4OAc<br/>
| <br/>
| 1 M
| <br/>
| 5
| 50 mM&nbsp;<br/>
|-
| MgSO4<br/>
| <br/>
| 0.1 M
| <br/>
| 10
| 10 mM&nbsp;<br/>
|-
| Triton X-100<br/>
| <br/>
| 10%
| <br/>
| 5
| 0.5%&nbsp;<br/>
|-
| '''BSA'''<br/>
| <br/>
| 10mg/ml
| <br/>
| 5
| 0.5 mg/mL&nbsp;<br/>
|-
| H<sub>2</sub>O
| <br/>
| <br/>
| <br/>
| 60
| <br/>
|}

'''&nbsp;5X HF Kfu Buffer&nbsp; 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<br/>
| 1.5 M, pH 10.0, adjust to pH 10 with KOH<br/>
| 75 ml<br/>
| 7.5 ml
| 10
| 150 mM<br/>
|-
| K2SO4
| Potassium sulfate, Sigma, ultrapure<br/>
| 0.5 M<br/>
| 200 ml
| 20 ml
| 40
| 200 mM
|-
| (NH<sub>4</sub>)<sub>2</sub>SO4<br/>
| Ammonium&nbsp;sulfate, J. T. Baker ultrapure<br/>
| 0.1 M<br/>
| 25 ml&nbsp;<br/>
| 62.5 ul of 4 M
| 5
| 5 mM&nbsp;<br/>
|-
| MgSO4<br/>
| Magnesium sulfate, Sigma, ultrapure<br/>
| 0.1 M<br/>
| 5 ml of 1 M<br/>
| 0.5 ml of 1 M
| 10
| 10 mM&nbsp;<br/>
|-
| Triton&nbsp;&nbsp;X-100<br/>
| Sigma,&nbsp;Ultrapure<br/>
| 10%<br/>
| 25 ml<br/>
| 2.5 ml 0f 10%
| 5
| 0.5%<br/>
|-
| H<sub>2</sub>O
| Water,&nbsp;DNase- RNase-free, Ultrapure<br/>
| <br/>
| &nbsp;ml&nbsp;<br/>
| 19.5 ml
| 30
| <br/>
|}

'''5X&nbsp; &nbsp;Kfu Buffer Recipe 3, the same as 2'''<br/>&nbsp;

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Name
| Source<br/>
| Stock<br/>
| 500 ml
| 100 ul
| Final<br/>
|-
| Tris<br/>
| <br/>
| 1.5M
| <br/>
| 10
| 150 mM , pH 10<br/>
|-
| K2SO4<br/>
| <br/>
| 0.5 M
| <br/>
| 40
| 200 mM&nbsp;<br/>
|-
| (NH<sub>4</sub>)<sub>2</sub>SO4<br/>
| <br/>
| 0.1 M
| <br/>
| 5
| 5 mM&nbsp;<br/>
|-
| MgSO4<br/>
| <br/>
| 0.1 M
| <br/>
| 10
| 10 mM&nbsp;<br/>
|-
| Triton X-100<br/>
| <br/>
| 10%
| <br/>
| 5
| 0.5%&nbsp;<br/>
|-
| H<sub>2</sub>O
| <br/>
| <br/>
| <br/>
| 30
| <br/>
|}







'''5X&nbsp; &nbsp;Kfu Buffer&nbsp; 4,&nbsp; similar to 1,&nbsp; with 1 M trehalose'''



{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Name
| Source<br/>
| Stock<br/>
| 500 ml
| 100 ul
| Final<br/>
|-
| Tris<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| 150 mM , pH 10<br/>
|-
| KCl<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| 50 mM&nbsp;<br/>
|-
| NH4OAc<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| 50 mM&nbsp;<br/>
|-
| MgSO4<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| 10 mM&nbsp;<br/>
|-
| Triton X-100<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| 0.5%&nbsp;<br/>
|-
| BSA<br/>
| <br/>
| <br/>
| <br/>
| <br/>
| 0.5 mg/mL&nbsp;<br/>
|-
| '''Trehalose'''
| <br/>
| <br/>
| <br/>
| <br/>
| 1 M
|-
| H<sub>2</sub>O
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|}

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

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| Name
| Source<br/>
| Stock<br/>
| 500 ml
| 100 ul
| Final<br/>
|-
| Tris<s>-HCl</s><br/>
| <br/>
| 1.5 M
| <br/>
| <br/>
| 100 mM , pH <s>8.8 </s>, 10<br/>
|-
| KCl
| <br/>
| 1 M
| <br/>
| <br/>
| 50 mM&nbsp;<br/>
|-
| (NH<sub>4</sub>)<sub>2</sub>SO4<br/>
| <br/>
| 0.1 M
| <br/>
| <br/>
| 50 mM&nbsp;<br/>
|-
| MgSO4<br/>
| <br/>
| 0.1 M
| <br/>
| <br/>
| 10 mM&nbsp;<br/>
|-
| Triton X-100<br/>
| <br/>
| 10%
| <br/>
| <br/>
| 0.5%&nbsp;<br/>
|-
| H<sub>2</sub>O
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|}



Buffer 6, Pfu buffer from Promega

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| Name
| Source<br/>
| Stock<br/>
| 500 ml
| 100 ul
| Final<br/>
|-
| Tris<s>-HCl</s><br/>
| <br/>
| 1.5 M
| <br/>
| <br/>
| 100 mM , pH <s>8.8 </s>, 10<br/>
|-
| KCl
| <br/>
| 1 M
| <br/>
| <br/>
| 50 mM&nbsp;<br/>
|-
| (NH<sub>4</sub>)<sub>2</sub>SO4<br/>
| <br/>
| 0.1 M
| <br/>
| <br/>
| 50 mM&nbsp;<br/>
|-
| MgSO4<br/>
| <br/>
| 0.1 M
| <br/>
| <br/>
| 10 mM&nbsp;<br/>
|-
| Triton X-100<br/>
| <br/>
| 10%
| <br/>
| <br/>
| 0.5%&nbsp;<br/>
|-
| BSA
| <br/>
| 10 mg/ml
| <br/>
| <br/>
| 0.5 mg/ml
|-
| H<sub>2</sub>O
| <br/>
| <br/>
| <br/>
| <br/>
| <br/>
|}



== Experiement ==

PCR: 5 ul of each buffer, 1 ul of 10 mM dNTP,&nbsp;1 ul of 10 uM 986-F1,&nbsp;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&nbsp;

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

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

[[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&nbsp;

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

Enzyme Prep: add 20 ul of 10 mg/ml BSA, 10 ul 1 M DTT, 12 ul of 1 mg/ml of Kfu&nbsp;to&nbsp; storage buffer:&nbsp;20 mM HEPES&nbsp;(pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl,&nbsp; &nbsp;and 50&nbsp;% 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<br/>500mM Sarcosine<br/>400mM KCl<br/>80mM (NH4)2SO4<br/>50mM Arginine<br/>50mM POPSO<br/>7.5mM MgCl2<br/>0.5% Tween-20<br/>5(-25%) Glycerol (depending on GC)<br/>5(-15%) DMSO (depending on GC)<br/>pH = 8.8

<br/>Additional components for Green Buffer (for direct Gel loading);<br/><12.5% (w/v) Ficoll 400 (as low as possible whilst still allowing for crisp bands)<br/>0.03% (w/v) Tartrazine<br/>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/]<br/>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:&nbsp;2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 ug/ml BSA ,&nbsp; stable at 20 C for at least 3 months,&nbsp;[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'''

&nbsp;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