Colony PCR - Revision history

ESO wikiadmin at 16:39, 29 May 2025

2025-05-29T16:39:46Z

← Older revision		Revision as of 18:39, 29 May 2025
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	Thermocycler Protocol		== Thermocycler Protocol ==

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	\|1		\|1
	\|Innitial melting		\|Innitial melting

ESO wikiadmin at 16:38, 29 May 2025

2025-05-29T16:38:05Z

← Older revision		Revision as of 18:38, 29 May 2025
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	== Reaction mix (Pipett scheme) ==		{{Minimal-Box
	H<sub>2</sub>O 16.5 μL		\|titel=<div>Reaction mix (Pipett scheme)</div>
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			<div>H<sub>2</sub>O 16.5 μL

	10x Taq/Pfu buffer 2 μL		10x Taq/Pfu buffer 2 μL

	Primer 1 0.4 μL (10 pmol)		Primer 1 0.4 μL (10 pmol)
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	dNTP-Mix 0.2 μL		dNTP-Mix 0.2 μL

	Taq polymerase 0.5 μL total 20 μL		Taq polymerase 0.5 μL total
			<hr style="border: 1px solid #333; margin: 20px 0;">
			20 μL
			</div>
			}}

			Thermocycler Protocol

	{\| class="wikitable"		{\| class="wikitable"

ESO wikiadmin at 14:10, 29 May 2025

2025-05-29T14:10:37Z

← Older revision		Revision as of 16:10, 29 May 2025
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	Reaction mix:		== Reaction mix (Pipett scheme) ==

	H<sub>2</sub>O 16.5 μL		H<sub>2</sub>O 16.5 μL

ESO wikiadmin: Created page with "Reaction mix: H₂O 16.5 μL 10x Taq/Pfu buffer 2 μL Primer 1 0.4 μL (10 pmol) Primer 2 0.4 μL (10 pmol) dNTP-Mix 0.2 μL Taq polymerase 0.5 μL total ..."

2025-05-29T14:10:19Z

Created page with "Reaction mix: H<sub>2</sub>O 16.5 μL 10x Taq/Pfu buffer 2 μL Primer 1 0.4 μL (10 pmol) Primer 2 0.4 μL (10 pmol) dNTP-Mix 0.2 μL Taq polymerase 0.5 μL total ..."

New page

Reaction mix:

H<sub>2</sub>O 16.5 μL

10x Taq/Pfu buffer 2 μL

Primer 1    0.4 μL (10 pmol)

Primer 2    0.4 μL (10 pmol)

dNTP-Mix    0.2 μL

Taq polymerase    0.5 μL total 20 μL

{| class="wikitable"
|1
|Innitial melting
|94 ºC
|3 minutes
|-
| colspan="4" |''Start-Loop (20x Cycles)''
|-
| colspan="2" |2    Denaturation
|94 ºC
|20 seconds
|-
| colspan="2" |3    Hybridization
|58 ºC
|20 seconds
|-
| colspan="2" |4    Elongation
|72 ºC
|60 seconds per 1000 bp
|-
| colspan="4" |''Close-Loop''
|-
|5
|Elongation
|72 ºC
|5 min
|-
|6
|Stop
|8 ºC
|
|}

== Procedure ==

* Prepare a “Chess board” LB-plate of corresponding resistance
* For each colony:
** lightly touch the colony with a yellow pipett-tip, on a 20µl pipett
** Touch a numbered chess-board-tile with a pipett-tip (3 times)
** Place the same tip in a corresponding filled PCR-reaction tube a resuspend 2-3 times
* Run PCR and incubate LB-Plate on 37°C
* Analyse PCR via Gel electrophoresis and identify positive clones
* On the same day positive clones can be expanded from the “Chess board”-LB onto a whole new plate (even if colonies are not yet visible)

Colony PCR - Revision history

ESO wikiadmin at 16:39, 29 May 2025

ESO wikiadmin at 16:38, 29 May 2025

ESO wikiadmin at 14:10, 29 May 2025

ESO wikiadmin: Created page with "Reaction mix: H2O 16.5 μL 10x Taq/Pfu buffer 2 μL Primer 1 0.4 μL (10 pmol) Primer 2 0.4 μL (10 pmol) dNTP-Mix 0.2 μL Taq polymerase 0.5 μL total ..."

ESO wikiadmin: Created page with "Reaction mix: H₂O 16.5 μL 10x Taq/Pfu buffer 2 μL Primer 1 0.4 μL (10 pmol) Primer 2 0.4 μL (10 pmol) dNTP-Mix 0.2 μL Taq polymerase 0.5 μL total ..."