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From 2010.igem.org

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Revision as of 19:24, 29 July 2010

Contents 1 Andreas 1.1 LMWP ligation 1.1.1 LMWP primer dilutions 1.1.2 Ligation

Andreas

LMWP ligation

Nina back from vacation and we decided to start working harder on making our CPPs. We divided TAT, Transportan10 and LMWP between us; I got LMWP.

LMWP primer dilutions

All LMWP primers were diluted 100x and 1000x, to yield concentrations of ≈1.0 and 0.1 μM, respectively:

Primer	Original conc.	100x dil. conc.	1000x dil. conc.
LMWP_C_F1	109 mM	1.09 mM	0.11 mM
LMWP_N_F1	141 mM	1.41 mM	0.14 mM
LMWP_CN_R1	131 mM	1.31 mM	0.13 mM
LMWP_CN_F2	120 mM	1.20 mM	0.12 mM
LMWP_C_R2	119 mM	1.19 mM	0.12 mM
LMWP_N_R2	139 mM	1.39 mM	0.14 mM

Flanking primers are designed with EcoRI and PstI overhangs, allowing me to use the linearized pSB1C3 vector for the ligation reaction.

Ligation

LMWP designed in two versions:

• C-LMWP containing the standard Freiburg prefix.
• N-LMWP containing the Freiburg N-part prefix.

Two ligation reactions performed for each LMWP version: (1) with high concentration primers (100x dil.) and (2) with low concentration primers (1000x dil.).

Ligation mixes

Ingredient	C-LMWP (high conc.) [μl]	N-LMWP (high conc.) [μl]	C-LMWP (low conc.) [μl]	N-LMWP (low conc.) [μl]
pSB1C3 vector (25 ng/μl)	2	2	2	2
LMWP_C_F1	1	0	1	0
LMWP_CN_R1	1	1	1	1
LMWP_CN_F2	1	1	1	1
LMWP_C_R2	1	0	1	0
LMWP_N_R2	0	1	0	1
2X Quick Ligase buffer	10	10	10	10
dH2O	3	3	3	3
Quick Ligase (added later)	1	1	1	1
Total volume	20	20	20	20

Procedures

1. Denaturation: 95°C, 10 min