Melbourne/Lab Notebook gv 5

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< Melbourne(Difference between revisions)
 
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{| border="1"
{| border="1"
-
|+ Site dirrected Mutagenesis round #4
+
|+ Site directed Mutagenesis round #3
|-
|-
!Mutation Number !!Template DNA (10ng)!! Sence Primer !! Antisence Primer !!#Colonies !! Picks named
!Mutation Number !!Template DNA (10ng)!! Sence Primer !! Antisence Primer !!#Colonies !! Picks named
|-
|-
-
!21|| 5A||  GvpL-g351a || GvpL-g351a-R || 21A,21B||  
+
!41|| 31A||  GvpL-g213a || GvpL-g213a-R || lots || 41A,41B,41C
|-
|-
-
!22|| 6A ||  GvpL-g318a || GvpL-g318a-R || 3|| 22A,22B,22C
+
!42|| 42A||  GvpL-g213a  || GvpL-g213a-R || lots || 42A,42B
|-
|-
-
!23|| 11A ||  GvpQ-g183a || GvpQ-g183a-R || 26|| 23A,23B
+
!43|| 32B||  GvpL-g696a  || GvpL-g696a-R || lots || 43A,43B
|-
|-
-
!24|| 10B ||  GvpP-g441a || GvpP-g441a-R || 60|| 24A,24B
+
!44|| 33A ||  GvpA-t57c || GvpA-t57c-R || lots || 44A,44B
-
|-
+
-
!25|| 6A || GvpL-g696a || GvpL-g696a-R || 1|| 25A,25B  (new PCR conditions)
+
-
|-
+
-
!26|| 6A ||  GvpL-g213a  || GvpL-g213a-R || || 26A,26B  (new PCR conditions)
+
-
|-
+
-
!27|| 10B ||  GvpQ-g150a || GvpQ-g150a-R  || || 27A,27B (new PCR conditions)
+
-
|-
+
-
!28|| 10B ||  GvpQ-g150a || GvpQ-g150a-R || None||
+
|}
|}
-
*Ran gel of 10uL sample of pcr reaction with 1uL of bluejuice.  [[Image:Melbourne-gel of pcr.jpg|right|thumb|300px|PCR product run on gel]]
+
 
-
** showed lane GvpQ-g150a non functional in old conditions but functional in new conditions.
+
* Picked colonies from Plates and [[Melb:Growing up cells|culture overnight ]]
* Picked colonies from Plates and [[Melb:Growing up cells|culture overnight ]]
* [[Melbourne/Glycerol Stocks|Produced glycerol stocks]] from 900uL of each overnight culture.
* [[Melbourne/Glycerol Stocks|Produced glycerol stocks]] from 900uL of each overnight culture.
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|+ DNA concentrations in ng/uL
|+ DNA concentrations in ng/uL
|-
|-
-
!History!!Mutation tube\colony: !! A !! B !!C !!D !!E
+
!History!!Mutation tube\colony: !! A !! B !! C
|-
|-
-
!pNL29T1-(Comp-g318a)-(GvpL-g351a)->!!21|| 183 
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!41|| 111 || 115 || 129
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g318a)->!!22|| 176
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!42|| 133 || 129
|-
|-
-
!pNL26P3-(GvpP-g441a)-(GvpQ-g183a)->!!23|| 247
+
!pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->!!43|| 107 || 75
|-
|-
-
!pNL26P3-(GvpQ-g183a)-(GvpP-g441a)->!!24||
+
!pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->!!44|| 221 || 204
-
|-
+
-
!pNL29T1-(GvpL-g351a)-(GvpL-g696a)->!!25||
+
-
|-
+
-
!pNL29T1-(GvpL-g351a)-(GvpL-g213a)->!!26||
+
-
|-
+
-
!pNL26P3-(GvpQ-g183a)-(GvpQ-g150a)->!!27||  
+
|}
|}
* Diagnostic digests were performed using
* Diagnostic digests were performed using
-
**A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 2h30min. (25uL reaction)
+
**A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 2hrs. (25uL reaction)
-
**B) 21.5uL of each p29T1 based colony in Buffer2 with 1uL EcoRI (20U) and 1uL BamHI (20U)  at 37degC for 2h30min. (26uL reaction)
+
**B) 5uL   of each in Buffer2 with 1uL EcoRI (20U) and 1uL BamHI (20U)  with 16.5uL of milliQ at 37degC for 2h. (26uL reaction) except pNL26 based 44A,B which were digested with EcoRI only.
-
**C) 21.5uL of each pNL26P3 based colony and in Buffer2 with 1uL EcoRI (20U) at 37degC for 2h30min. (25uL reaction)
+
**C) 10  uL of each in Buffer2 with 2uL HindIII (20U) with 11.5 uL milliQ at 37degC for 1hour. (25uL reaction)
-
* Prepared two 20 lane 100mL [[Melbourne/Preparing an agarose gel|agarose gel]]
+
* Prepared one 2row x 20 lane gel 100mL [[Melbourne/Preparing an agarose gel|agarose gel]]
*[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest  
*[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest  
*Digest Pattern
*Digest Pattern
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!History!!Mutation tube !!FRAGMENTS  
!History!!Mutation tube !!FRAGMENTS  
|-
|-
-
!pNL29T1-(Comp-g318a)-(GvpL-g351a)->!!21|| || 5983|| 2516!! 483|| <-  
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!41|| !! 8982!! <-
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g318a)->!!22|| || 5983|| 2516!! 483|| <-
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!42|| !! 8982!! <-
|-
|-
-
!pNL26P3-(GvpP-g441a)-(GvpQ-g183a)->!!23 || !! 4148 || 3170 || 2516 || 378 !! <- || 105
+
!pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->!!43|| !! 8982!! <-
|-
|-
-
!pNL26P3-(GvpQ-g183a)-(GvpP-g441a)->!!24 || !! 4148 || 3170 || 2516 || 378 !! <- || 105  
+
!pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->!!44|| !! 7318 || 2516 || 378 || 105  
 +
|}
 +
*Results of PstI diagnostics:[[Image:Melbourne-gv mut 4p.jpg|left|thumb|850px|PstI digest]]
 +
 
 +
{| border="1"
 +
|+ EcoRI and BamHI digest (44 with EcoRI only)
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g696a)->!!25|| || 5983!! 2894|| <-|| 105
+
!History!!Mutation tube !!FRAGMENTS
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g213a)->!!26 || !!6088|| 2516|| 378|| <-
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!41|| || 6871|| 2112
|-
|-
-
!pNL26P3-(GvpQ-g183a)-(GvpQ-g150a)->!!27 || || 3928 !! 3390 || 2516 || 378 !! <- || 105
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!42|| || 6871|| 2112
 +
|-
 +
!pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->!!43|| || 6871|| 2112
 +
|-
 +
!pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->!!44  || !! 10318 || <-
|}
|}
-
*Results of PstI diagnostics:[[Image:Melbourne-gv mut 2p.jpg|right|thumb|850px|PstI digest]]
+
 
-
[[Image:Melbourne-gv mut 2e.jpg|left|thumb|350px|EcoRI & BamHI digests]]
+
{| border="1"
{| border="1"
-
|+ EcoRI and BamHI digest
+
|+ HindIII digest
|-
|-
-
!History!!Mutation tube!!Fragments: 
+
!History!!Mutation tube !!FRAGMENTS
|-
|-
-
!pNL29T1-(Comp-g318a)-(GvpL-g351a)->!!21|| !! 6871|| 2112!! <-
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!41|| || 4852|| 3479 || 652
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g318a)->!!22||  !! 6871|| 2112!! <-
+
!pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->!!42||  || 4852|| 3479 || 652
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g696a)->!!25|| !! 6871|| 2112!! <-
+
!pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->!!43|| || 4852|| 3479 || 652
|-
|-
-
!pNL29T1-(GvpL-g351a)-(GvpL-g213a)->!!26|| !! 6871|| 2112!! <-
+
!pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->!!44  || || 4852|| 3479 || 1335 || 652
|}
|}
-
 
+
[[Image:Melbourne-gv mut 4e.jpg|left|thumb|850px|EcoRI / BamHI digests and HindIII digest]]
-
*Results of EcoRI diagnostics:
+
*Results of digests: All look good except 44B which is still being cut ecoRI at t57c (background incomplete digestion of template using dpnI). There does however appear to be a small variation in size but it is hard to tell if this is gel or concentration inconsistancy. 41C HindII 3479 band appears slightly larger than 41A or B.
-
Conclude 21A,22A,23A are all satisfactory.
+

Latest revision as of 02:40, 26 October 2007

<Return to lab book summary>

Prepared for site dirrected mutagenesis

  • Diluted DNA from last round, creating 10ng/ul sample and then adding 1uL of this to 36uL milliQ water to produce required 10ng/37uL template for PCR.
Tube conc of miniprep ng/ul total=(100ul) 1uL miniprep added to x uL milliQ -> 10ng/uL
31A 117 10.7
31B 133 12.3
32B 96 8.6
33A 114 10.4

Site dirrected Mutagenesis Round #4

  • Applied the stratagene Site directed mutagenesis protocolto the following DNA and primer pairs, which when plated out on LB AMP plates producing the numbers of colonies shown. Several of these were picked and tubes marked with a character suffix as shown in table.


Site directed Mutagenesis round #3
Mutation Number Template DNA (10ng) Sence Primer Antisence Primer #Colonies Picks named
41 31A GvpL-g213a GvpL-g213a-R lots 41A,41B,41C
42 42A GvpL-g213a GvpL-g213a-R lots 42A,42B
43 32B GvpL-g696a GvpL-g696a-R lots 43A,43B
44 33A GvpA-t57c GvpA-t57c-R lots 44A,44B
DNA concentrations in ng/uL
HistoryMutation tube\colony: A B C
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->41 111 115 129
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->42 133 129
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->43 107 75
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->44 221 204
  • Diagnostic digests were performed using
    • A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 2hrs. (25uL reaction)
    • B) 5uL of each in Buffer2 with 1uL EcoRI (20U) and 1uL BamHI (20U) with 16.5uL of milliQ at 37degC for 2h. (26uL reaction) except pNL26 based 44A,B which were digested with EcoRI only.
    • C) 10 uL of each in Buffer2 with 2uL HindIII (20U) with 11.5 uL milliQ at 37degC for 1hour. (25uL reaction)
  • Prepared one 2row x 20 lane gel 100mL agarose gel
  • Loaded 20uL of digest
  • Digest Pattern
  • Expected effects
PstI digest
HistoryMutation tube FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->41 8982 <-
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->42 8982 <-
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->43 8982 <-
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->44 7318 2516 378 105
  • Results of PstI diagnostics:
    PstI digest
EcoRI and BamHI digest (44 with EcoRI only)
HistoryMutation tube FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->41 6871 2112
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->42 6871 2112
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->43 6871 2112
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->44 10318 <-
HindIII digest
HistoryMutation tube FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->41 4852 3479 652
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)->42 4852 3479 652
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)->43 4852 3479 652
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)->44 4852 3479 1335 652
EcoRI / BamHI digests and HindIII digest
  • Results of digests: All look good except 44B which is still being cut ecoRI at t57c (background incomplete digestion of template using dpnI). There does however appear to be a small variation in size but it is hard to tell if this is gel or concentration inconsistancy. 41C HindII 3479 band appears slightly larger than 41A or B.
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