"

From 2010.igem.org

Revision as of 17:50, 26 October 2010 (view source) Jesús (Talk | contribs) ← Older edit		Latest revision as of 06:41, 27 October 2010 (view source) Jesús (Talk | contribs) (→Wenesday)
(10 intermediate revisions not shown)
Line 10:		Line 10:
	==''Monday''==		==''Monday''==
-	==='''Well...we are stuck with the vector this week we have to get vector is our main objetive.'''===	+	==='''Well...we are stuck with the vector. This week we have to get it.'''===
-	*'''We have achived a nanodrop readings as below'''	+	*'''We have achieved a nano spectrophotometer and readings are below:'''
Line 73:		Line 73:
-	*'''Yep our trouble is the lisis alcaline method to do the miniprep'''	+	*'''Yep, our trouble is the miniprep method'''
-	'''we have a low concentration of vector’s plasmid.'''	+	'''we have a low concentration of vector plasmid.'''
-	'''We have to work an try to get more plasmid and make dilutions of PCR’s'''	+	'''We have to work an try to get more plasmid and used diluted PCR products'''
-	'''is not posible to do ligations with this ratio between vector an insert.'''	+	'''is not possible achieve ligations with this vector an insert ratio.'''
-	'''On this step we advisor has proposed a method via Low Meelting agarose'''	+	'''Our advisor proposed use low Melting agarose'''
-	'''to geting out plasmid from the band.'''	+	'''to extract the plasmid from the gel.'''
	==''Tuesday''==		==''Tuesday''==
-	==='''We run with a low meelting's agarose prove to extract band tomorrow'''===	+	==='''We ran with a low meelting agarose and cut off the band '''===
-	==='''we do a protocol to precipit with alchol an salts our objetive is do it all'''===	+	==='''we precipitated using alchol and salts in order '''===
-	==='''to get vector and do the ligations.'''===	+	==='''to get enough vector to do the ligations.'''===
Line 97:		Line 97:
-	==''Wensday''==	+	==''Wednesday''==
	==='''Plan'''===		==='''Plan'''===
-	*'''We prepared all to do ligations PsB1C3 with our Pcr's.'''	+	*'''We set up all to do the ligations.'''
-	*'''For this we going to cut with EcoRI and PstI both vector and insert.'''	+	*'''We digested both vector and insert with ''EcoR''I and ''Pst''I .'''
-	*'''We'll run a low melting gel slow 1 hour and an half 80 volts then purified from the band.'''	+	*'''We ran a low melting agarose gel 1 hour at 80 volts, then cut off the band from gel.'''
-	'''At Claudia's lab we cut tree bands from 2% agarose's gel of which two of'''	+	'''At Claudia's lab we tried three two different kits to recover DNA from gel.'''
-	'''them where purified by Axigen kit the third one by a diferent kit.'''	+	*'''Mobio ultra clean Microbial DNA kit'''
		+	*'''Axygen.....'''
		+	*'''
-	'''The digestion was done with following amounts.'''	+	'''The digestions were done with following amounts.'''
	{| border="1"		{| border="1"
Line 134:		Line 136:
	|30μl		|30μl
	|}		|}
-
	==''Thursday''==		==''Thursday''==
Line 140:		Line 141:
	==='''Plan'''===		==='''Plan'''===
-	'''We going to proceed to do a dilutions and do the ligation'''	+	'''We then proceed to diluted PCR fragments'''
-	'''For this we do the following accounts'''	+	'''For this we did the following operations:'''
	'''Poner las fórmulas usadas en un formato bonito'''		'''Poner las fórmulas usadas en un formato bonito'''
-	'''We did the ligations using the following amounts.'''	+	'''We did the ligations using the following amounts:'''
	{| border="1"		{| border="1"
Line 170:		Line 171:
-	'''We used the follow notation.'''	+	'''We used the follow denomination.'''
-	*'''For vector purified from band:               Pu'''	+	*'''Band purified vector :                             Pu'''
-	*'''For iGEM's vector (Vial with green cover):  P'''	+	*'''iGEM's vector (Vial with green cover):  P'''
-	*'''Vector  only digest:                         S'''	+	*'''Digested vector:                                       S'''
-	'''After one hour of ligation we transform by thermic shock'''	+	'''After one hour ligation we transformed by heat shock'''
-	'''and left overnight plates to 37º'''	+	'''and kept overnight the plates at 37ºC'''
Line 184:		Line 185:
	==''Friday''==		==''Friday''==
-	'''Today we discuss what we going to do with the AFP (anti freeze protein),'''	+	'''Today we discussed what we going to do with the AFP (anti freeze protein),'''
-	'''We going to transform by thermic shock plating on kanamicina medium then do'''	+	'''We decided transform by heat shock and plating it on kanamicina medium, then prepare'''
-	'''miniprep and cut with Xba and PstI we have to ligate at J13002 this as blackbone'''	+	'''miniprep plasmid and cut it with ''Xba''I and ''Pst''I.  We have to ligate it to the backbone  J13002'''
-	'''the blackbone will cut with SpeI and PstI.'''	+	'''the backbone had to be  cut with ''Spe''I and ''Pst''I.'''
-	'''Meanwhile our ligations'''	+	'''Meanwhile, here are the  previous day ligations.'''
-	*'''PCR1 Lig S'''	+	*'''PCR1 Lig S Positive!!!!!!!'''
-	*'''PCR2 Lig S'''	+	*'''PCR2 Lig S Positive!!!!'''
-	*'''PCR3 Lig S'''	+	*'''PCR3 Lig S Positive!!!!'''
-	*'''PCR4 Lig Pu'''	+	*'''PCR4 Lig Pu Positive!!!!'''
-	*'''PCR4 Lig S'''	+	*'''PCR4 Lig S Positive!!!!'''
	[[Image:Labo_254.JPG|400px]]		[[Image:Labo_254.JPG|400px]]

---

Latest revision as of 06:41, 27 October 2010

Week #4

27th September - 01 October 2010

Monday

Well...we are stuck with the vector. This week we have to get it.

• We have achieved a nano spectrophotometer and readings are below:

	ng/μl	260/280	260/230
PsB1C3	0.60	-0.73	-0.0
PCR 1 red	427.70	1.71	1.71
PCR 2 red	483.20	1.74	1.71
PCR 3 red	410.70	1.76	2.02
PCR 4 red	431.05	1.61	1.88
PCR 1 blue	533.35	1.71	1.75
PCR 2 blue	536.00	1.72	1.82
PCR 3 blue	577.50	1.69	1.59
PCR 4 blue	627.55	1.70	1.56
PsB1C3	4.10	2.62	1.01

• Yep, our trouble is the miniprep method

we have a low concentration of vector plasmid.

We have to work an try to get more plasmid and used diluted PCR products

is not possible achieve ligations with this vector an insert ratio.

Our advisor proposed use low Melting agarose

to extract the plasmid from the gel.

Tuesday

We ran with a low meelting agarose and cut off the band

we precipitated using alchol and salts in order

to get enough vector to do the ligations.

Wednesday

Plan

• We set up all to do the ligations.
• We digested both vector and insert with EcoRI and PstI .
• We ran a low melting agarose gel 1 hour at 80 volts, then cut off the band from gel.

At Claudia's lab we tried three two different kits to recover DNA from gel.

• Mobio ultra clean Microbial DNA kit
• Axygen.....

The digestions were done with following amounts.

DNA	20μl
Buffer NB2	3μl
EcoRI	2μl
PstI	2μl
H2O	2.4μl
BSA	0.6μl
Total	30μl

Thursday

Plan

We then proceed to diluted PCR fragments

For this we did the following operations:

Poner las fórmulas usadas en un formato bonito

We did the ligations using the following amounts:

Insert [20ng/μl]	5μl
Vector	1μl
Reaction buffer	1μl
T4 DNA Ligase	1μl
H2O	2μl
Total	10μl

We used the follow denomination.

• Band purified vector : Pu
• iGEM's vector (Vial with green cover): P
• Digested vector: S

After one hour ligation we transformed by heat shock

and kept overnight the plates at 37ºC

Friday

Today we discussed what we going to do with the AFP (anti freeze protein),

We decided transform by heat shock and plating it on kanamicina medium, then prepare

miniprep plasmid and cut it with XbaI and PstI. We have to ligate it to the backbone J13002

the backbone had to be cut with SpeI and PstI.

Meanwhile, here are the previous day ligations.

• PCR1 Lig S Positive!!!!!!!
• PCR2 Lig S Positive!!!!
• PCR3 Lig S Positive!!!!
• PCR4 Lig Pu Positive!!!!
• PCR4 Lig S Positive!!!!