IGEM:Groningen/Notebook/iGEM 2011/2011/07/20

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20-7-11



Sequencing update:
So far the right constructs: PhybB-RBS-GFP-DT, PcI-RBS-GFP-DT, PlasI-RBS-GFP-DT, PhybB in pSB1C3, cI-LVA-DT.
Send for sequencing: PBAD-RBS-GFP-DT, PBAD-cI-LVA and today check PhybB-cI-LVA ( will be send tomorrow)
Sequencing failed: LasR-LVA, the beginning is not right, sequence conflict. On protein levels this is the case:
LasR-LVA as it should be: Met A L V D G F L E L E R S S G K L E W S A I L Q K Met A S D L G F S K I L F G L L P K D S Q D Y E
N A F I V G N Y P A A W R E H Y D R A G Y A R V D P T V S H C T Q S V L P I F W E P S I Y Q T R K Q H E F F E E A S A A G L V
Y G L T Met P L H G A R G E L G A L S L S V E A E N R A E A N R F I E S V L P T L W Met L K D Y A L Q S G A G L A F E H P V S
K P V V L T S R E K E V L Q W C A I G K T S W E I S V I C N C S E A N V N F H Met G N I R R K F G V T S R R V A A I Met A V N
L G L I T L Stop Stop

Protein sequence of sample in sequencing results:

I K Stop A Y H E A E F Q I K K I L S F R Stop G Stop F L E L E W S A I L Q K Met A S D L G F S K I L F G L L P K D S Q
D Y E N A F I V G N Y P A A W R E H Y D R A G Y A R V D P T V S H C T Q S V L P I F W E P S I Y Q T R K Q H E F F E E A S A A
G L V Y G L T Met P L H G A R G E L G A L S L S V E A E N R A E A N R F I E S V L P T L W Met L K D Y A L Q S G A G L A F
E H P V S K P V V L T S R E K E V L Q W C A I G K T S W E I S V I C N C S E A N V N F H Met G N I R R K F G V T S R R V A
A I Met A V N L G L I T L A A N D E N Y A L V A Stop
Stop Y Stop S Q A S N K T K G S V E R L G L S F Y L L F V G E R S L L
E S H W L T F G W A F L R L Y T S S G R C S P A K K G K V S P P C P F S L K P K D Y F A L C X F X A H Stop


<Colony PCR with samples PhybB-cI-LVA-DT (new transformants! Self ligation plate looked very well!)was done as followed:
Mastermix scheme:
Taq 10× buffer with NH4SO4 without MgCl2: 20.00μl
dNTPs 10mM each: 4μl
MgCl2: 12μl
Taq polymerase 5u/μl: 1μl
Biobrick vector forward primer 10μM: 4μl
Biobrick vector reverse primer 10μM: 4μl
MilliQ water: 155μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle (33×)
Denaturation: 94°C for 30s.
Annealing: 60°C for 30s.
Extension: 72°C for 2 min.
Final extension: 72°C for 10 min.
Store at 4°C infinite

PCR products were analysed on a 1% agarose gel with TBE.
PhybB-cI-LVA-DT can be right, is difficult to see, maybe use some colonies-> 4 were used for overnight culture so tomorrow a
plasmid prep should be done.

PCR with LasR-LVA:
Mastermix scheme:
10× pfu buffer with MgSO4: 5.00μl
dNTPs 10mM each: 1μl
Pfu polymerase 5u/μl: 1μl
Forward primer 10μM: 1μl
Reverse primer 10μM: 1μl
MilliQ water: 40μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 95°C for 3 min.
Cycle (35×)
Denaturation: 95°C for 30s.
Annealing: 60°C, 65°C and 70°C (gradient) for 30s.
Extension: 72°C for 2.5 min.
Final extension: 72°C for 15 min.
Store at 4°C infinite

PCR products were analysed on a 1% agarose gel with TBE.
PCR clean up+ measure DNA concentration with nanodrop.
PCR with BB primers: 125.3 ng/μl, PCR LasR-LVA primers: 89.1μl
Ligation calculator: need 57ng of PCR product (BB) and 46ng of PCR product (LasR-LVA primers)
After calculation:
Need 3.5μl PCR product (BB) for digestion and 6.5μl of the clean up for ligation. Need 4μl of PCR product (LasR-LVA primers)
and 6.5μl for ligation
Digestion:
LasR-LVA
3.5 and 4μl insert
1μl SpeI
2μl Tango buffer
13.5 or 13μl MQ
Total volume (V): 20μl
After 1.5 hours incubation:
1μl EcoRI (NORMAL one NOT fast digest!) and (20/8:) 2.5 μl Tango buffer
after half an hour: start digesting the vector:
3μl vector
1μl EcoRI (fast digest)
1μl XbaI (fast digest)
3μl Fast digest buffer
1μl Fast AP
22μl MQ

Ligation: (overnight)

LasR-LVA in pSB1A3-DT
6.5μl insert
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
2μl MQ

Self ligation:
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl MQ
Ligate overnight in the fridge (at 4°C)

Colony PCR of today was not alright, compare with yesterday's-> yesterday samples should be OK, so plasmid prep and send for
sequencing




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