"

From 2012.igem.org

Revision as of 22:37, 13 July 2012 (view source) Gutiloluis (Talk | contribs) ← Older edit		Revision as of 23:02, 13 July 2012 (view source) Gutiloluis (Talk | contribs) (→Bacterial DNA extraction protocol) Newer edit →
Line 2:		Line 2:
	==='''Bacterial DNA extraction protocol'''===		==='''Bacterial DNA extraction protocol'''===
		+
		+	#Shaking culture overnight.
		+	#Centrifuge twice at 10,700 rpm  in 1.5 ml eppendorf tube 2 or 3 minutes. On the same tube centrifuge all the cultures and remove the supernatant.
		+	#Wash the pellet twice with 1 ml of NaCl 1M and Resuspend.
		+	#Centrifuge 2 minutes at 10,700 rpm and remove the supernatant.
		+	#Resuspend the pellet on 567 uL
	==='''Miniprep'''===		==='''Miniprep'''===

---

Revision as of 23:02, 13 July 2012

Contents 1 Protocols 1.1 Bacterial DNA extraction protocol 1.2 Miniprep 1.3 Electroporation 1.4 Primer Design 1.5 PCR - Pfu DNA polimerase 1.6 PCR - Taq DNA polimerase 1.7 PCR - Colony 1.8 PCR - Boiling

Protocols

Bacterial DNA extraction protocol

1. Shaking culture overnight.
2. Centrifuge twice at 10,700 rpm in 1.5 ml eppendorf tube 2 or 3 minutes. On the same tube centrifuge all the cultures and remove the supernatant.
3. Wash the pellet twice with 1 ml of NaCl 1M and Resuspend.
4. Centrifuge 2 minutes at 10,700 rpm and remove the supernatant.
5. Resuspend the pellet on 567 uL

Miniprep

Electroporation

Electro-competent cells of E. coli DH5α are electroporated at 1.25 mV, immediately we add SOC medium to the bacteria and we incubated on a shaker at 37°C for one hour, then, we culture cells in a solid medium containing the antibiotic of selection.

Primer Design

PCR - Pfu DNA polimerase

One reaction.

Reactives	Volume(μL)
H2O	17.95
Buffer	2
MgSO4	1.6
dNTP's	0.4
Primer Fw	0.4
Primer Rv	0.4
Taq	0.06
Pfu	0.19
DNA	2
Total	25

PCR - Taq DNA polimerase

One reaction.

Reactives	Volume(μL)
H2O	6.15
Buffer	1
MgCl2	0.5
dNTP's	0.25
Primer Fw	0.5
Primer Rv	0.5
Taq	0.1
DNA	1
Total	10

If needed add 1 μL of DMSO per reaction, and adjust the amount of H₂O to a final volume of 10 μL.

PCR - Colony

Based on the protocol used with Taq polimerase, add 1 μL more of H₂O and take a few cells from the strain pricking it with a toothpick and mixing them with the other PCR reactives instead of using purified DNA.

PCR - Boiling

We make a lysate of the cells and add 1 μL of that lysate instead of purified DNA using the Taq polimerase protocol. To obtain the lysate, scrape the strain and resuspend the cells taken on 50 μL of H₂O, then, put at 95°C for 10 minutes.