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From 2010.igem.org

Contents 1 Andreas 1.1 LMWP ligation 1.1.1 LMWP primer dilutions 1.1.2 Ligation 1.2 Colony PCR verification of pSB1x3 constructs from 13/7 colonies 1.2.1 Colony PCR 1.2.2 Gel verification 1.2.2.1 Results 1.2.3 ON cultures of verified clones

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
2. Temperature slowly lowered to 65°C
3. Annealing: 65°C, 5 min
4. Temperature slowly lowered to RT (≈27°C)
5. Ligation: Quick Ligase added
   • 25°C, 10 min
6. Transformation: 6 μl ligation mix into Top10. Cells grown on LB agar + 25 μg/ml Cm.

Colony PCR verification of pSB1x3 constructs from 13/7 colonies

pSBX verification primers VF2 and VR arrived. Decided to verify clones from 13/7 by colony PCR.

Colony PCR

From 13/7 colonies, pSB1x3

Picked new colonies from LB agar plates. Since the plates had now been left a few days in 4°C, the white (positive) clones were easier to discern from the red, as the red colonies now had a deeper red color.

Picked colonies were dissolved in 10 μl LB:

• pSB1C3.BBa_J18930 A
• pSB1C3.BBa_J18930 B
• pSB1C3.BBa_J18931 A
• pSB1C3.BBa_J18931 B
• pSB1C3.BBa_J18932 A
• pSB1C3.BBa_J18932 B
• pSB1C3.SOD A
• pSB1C3.SOD B
• pSB1C3.yCCS A
• pSB1C3.yCCS B

I decided not to pick the pSB1K3 and pSB1A3 constructs, as it is better to first verify single clones in one vector, and later transfer them to other vectors when needed.

PCR tubes - illustra PuReTaq Ready-To-Go™ PCR Beads:

• 1 μl 10 μM VF2 forward primer
• 1 μl 10 μM VR reverse primer
• 22.5 μl dH₂O
• 0.5 μl cell suspension
  • Positive control: 0.5 μl pSB1A3.BBa_J04450, pure plasmid
  • Negative control: 0.5 μl dH₂O

PCR settings

1. Denaturation: 95°C - 10:00
2. Denaturation: 95°C - 01:00
3. Annealing: 55°C - 00:30
4. Elongation: 72°C - 01:30
   • Cycle back to step 2 29 times
5. Elongation: 72°C - 10:00
6. 15°C - ∞

Gel verification

1 % agarose, 90 V, 40 min

Results

Expected sizes (including ≈300 bp from vector):

• BBa_J18930: 1040 bp
• BBa_J18931: 1040 bp
• BBa_J18932: 1030 bp
• SOD: 795 bp
• yCCS: 1085 bp
• BBa_J04450: 1400 bp

Results correspond well to expected fragment sizes (see attached gel image).

ON cultures of verified clones

ON cultures (5 ml + 25 μg/ml Cm) were set for plasmid prep. and glycerol stock prep.