Team:TU Delft/17 August 2010 content

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(Digestion)
(Ligation Kinetics)
 
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The digestion mix was incubated at 37 degrees for 1 hour and at 80 degrees for 20 minutes to inactivate the restriction enzymes. The mixes were checked on [http://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel 1% agarose gel]:
The digestion mix was incubated at 37 degrees for 1 hour and at 80 degrees for 20 minutes to inactivate the restriction enzymes. The mixes were checked on [http://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel 1% agarose gel]:
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[[Image:digestions_pSB1C3_18-08-2010.png|400px|thumb|left|1% agarose gel run at 90V for 50 min. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker]]
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[[Image:digestions_pSB1C3_18-08-2010.png|100px|thumb|left|1% agarose gel run at 90V for 50 min. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker]]
Lane description
Lane description
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|}
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From the gel it could be concluded that the digestion were succesful.
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From the gel it could be concluded that the digestions were succesful.
====Ligation====
====Ligation====
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Following the digestion the products were [[Team:TU_Delft/protocols/ligation|ligated]] for 15 minutes at RT:
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Following the digestion the products were [[Team:TU_Delft/protocols/ligation|ligated]] for 4 hours at 20 degrees and the ligase inactivated at 80 degrees for 20 min.:
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|None
|None
|‘E - pSB1C3 - P’
|‘E - pSB1C3 - P’
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|}
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 +
To test whether the ligations were succesful, 1 uL of ligation mix was used as template for a PCR amplification.
 +
 +
====Ligation Kinetics====
 +
 +
There are multiple protocols available describing ligation waiting times ranging from 30 minutes to 24 hours. It was decided to perform a ligation kinetics experiment. pSB1C3 was digested using EcoRI and PstI and checked on gel for complete digestion. NEB T4 ligase and ligase buffer were added to the mixture and the ligation mixes were incubated at 20 degrees as follows:
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 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''Incubation time [min]'''
|-
|-
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|7
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|1
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|014C
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|30
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|‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’
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|-
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|‘X - J61100-rubA4-J61100-rubR-B0015 - P’
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|2
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|‘E - pSB1C3 - P’ (Pre-shipped linearized plasmid)
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|60
 +
|-
 +
|3
 +
|120
 +
|-
 +
|4
 +
|240
|}
|}
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The ligations were performed in parallel: one batch using the NEB T4 DNA ligase and buffer, the other batch the Fermentas T4 DNA ligase and buffer. We hope to be able to discern the differences in efficiency between the two (if there are any).
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In essence this is the ligation of E-J04450-P back into E-pSB1C3-P. The number of red colonies on the CAM plates after transformation should be indicative of the ligation efficiency.
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To test whether the ligations were succesful, i uL of ligation mix was used as template for a PCR amplification. The results will be known by tomorrow.
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==Alkane Degradation parallel attempt==
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 +
====Transformation====
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The ligation products (10 ul) from yesterday were used to transform 30 uL of TOP10 cells.

Latest revision as of 08:27, 31 August 2010

Contents

Alkane degradation

Digestion

Yesterday's transformed competent cells of the 014C, 020C, 405C, 327C and 304C ligation mixes (using the BioBrick assembly method) yielded only red colonies, indicating the lack of positive colonies. It is interesting to note the presence of numerous red colonies on the digest control plate, whereas the ligation control plate (Digested pSB1C3 + ligase buffer + ligase) yielded but 1 red colony (at similar trasnsformation mix concentrations). Based on the information gathered thus far a few hypotheses were made:

Hypothesis I: The ligase buffer inhibits transformation efficiency in some way.

Hypothesis II: The ligation time of 20 mins was too short.

In order to test hypothesis I, the following mixes will be transformed into commercial competent cells:

# Fragment 1 Ligase buffer? Total volume (uL)
1 E-pSB1C3-P (2 uL) No 20
2 E-pSB1C3-P (2 uL) Yes (2 uL) 20

In order to test hypothesis II, a ligation kinetics assay was performed using E and P digested pSB1C3 as vector and J04450 as insert. The ligations were run for 30, 60, 120 and 240 minutes.

Furthermore, a second attempt was made at creating BioBricks 014C, 020C, 405C, 327C and 304C. pSB1C3 was digested additionaly using HindIII to cut the J04450 insert as follows:

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 pSB1C3 EcoRI PstI HindIII NEBuffer 3 ‘E - pSB1C3 - P’

The digestion mix was incubated at 37 degrees for 1 hour and at 80 degrees for 20 minutes to inactivate the restriction enzymes. The mixes were checked on 1% agarose gel:

1% agarose gel run at 90V for 50 min. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker

Lane description

# Description Expected size (bp) OK?
1 Smartladder Varies Yes
2 pSB1C3 E+P digest 2035, 1081 Yes
3 pSB1C3 E+P+HindIII digest 2035, 456, 403, 222 Yes

From the gel it could be concluded that the digestions were succesful.

Ligation

Following the digestion the products were ligated for 4 hours at 20 degrees and the ligase inactivated at 80 degrees for 20 min.:

# BioBrick Fragment 1 Fragment 2 Destination Vector
1 014C ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ ‘E - pSB1C3 - P’
2 020C ‘E - J23100-X-J61100-ladA - S’ ‘X - J61101-ADH - P’ ‘E - pSB1C3 - P’
3 405C ‘E - J23100-X-J61101-PhPFDα - S’ ‘X - J61101-PhPFDβ - P’ ‘E - pSB1C3 - P’
4 327C ‘E - pCaiF - S’ ‘X - B0032 - P’ ‘E - pSB1C3 - P’
5 304C ‘E - pAlkS - S’ ‘X - B0032 - P’ ‘E - pSB1C3 - P’
6 Ligation control None None ‘E - pSB1C3 - P’

To test whether the ligations were succesful, 1 uL of ligation mix was used as template for a PCR amplification.

Ligation Kinetics

There are multiple protocols available describing ligation waiting times ranging from 30 minutes to 24 hours. It was decided to perform a ligation kinetics experiment. pSB1C3 was digested using EcoRI and PstI and checked on gel for complete digestion. NEB T4 ligase and ligase buffer were added to the mixture and the ligation mixes were incubated at 20 degrees as follows:

# Incubation time [min]
1 30
2 60
3 120
4 240

In essence this is the ligation of E-J04450-P back into E-pSB1C3-P. The number of red colonies on the CAM plates after transformation should be indicative of the ligation efficiency.

Alkane Degradation parallel attempt

Transformation

The ligation products (10 ul) from yesterday were used to transform 30 uL of TOP10 cells.