IGEM:Peking/2007/Switch-Notebook/2007-8-12

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Contents

rescue by precipitation of fragment after digestion

electrophoresis result to show that SD2, SD3 and Prec successfully ligated.

Colony PCR of T1T2-pcc010

easy Taq 0.1uL

Pkan-F 0.5uL

pcc-test-R 0.5uL

Buffer 2uL

dNTP 2uL

ddH2O 15uL

We did choose some colonies that seems to be right according to the PCR band after electrophoresis.

Miniprep and double digestion test

T1T2-Pcc010

plasmid 3uL

SacII 0.5uL

T buffer 2uL

BSA 2uL

ddH2O 12.5uL

T1TE-T1T2-pcc010

plasmid 3uL

SalI 0.5uL

XhoI 0.5uL

H buffer 2uL

H2O 14uL

SD2 and SS2

plasmid 3uL

SalI 0.5uL

XhoI 0.5uL

H buffer 2uL

H2O 14uL

total 20uL system

re-PCR

lac, rec, sulA, SS1-3,SD1-3

Some of the fragment did appear this time after we change some data in PCR processing.

test of enzymatic sites on GFP-plx007

plasmid 10uL

XhoI/SalI 0.5uL each

buffer H 2uL

H2O 7uL

or

Plasmid 4uL

XbaI 0.5uL

buffer M 2uL

H2O 13.5uL

total 20uL system

ligation of SD2, SD3 and Prec with SDY-EGFP-plx007

ligation of SS1, SS2 and SS3 with SDY-EGFP-plx007

praparation of LB medium with Cm

miniprep of T1T2-pcc010

single digestion to test if the plasmid we isolated it right or not

digestion test of T1TE-T1T2-pcc010

plasmid 2uL

H buffer 10uL

Trition X-100 10uL

BSA 10uL

NotI/XhoI 2uL each

add ddH2O to a total of 100uL system

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