Team:Paris Bettencourt/Notebook/Riboswitch

July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

  1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
  2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
  3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
  4. Waiting 30 min in ice
  5. Heat shock 30 sec 42°C
  6. Then 5min in ice
  7. Add 200uL SOC medium
  8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
  9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control
    I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.


July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method
  • The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)
  • The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.
  • In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)
  • Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.
  • When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.
  • Centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.
  • Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.


    • July 30th

    Goal 1

    PCR my G block and backbone

    Procedure

  • PCR1 = Backbone + 068 and 069 primers, before purify
  • PCR2 = FINAL + 068 and 069 primers, before purify
  • Then PCR purification
  • Nanodrop : PCR 1 = 61ng/uL ; PCR2 = 25,4 ng/uL

    • Goal 2

    Digestion/Ligation/Transformation

    Procedure

    First, digestion :
  • PCR1 ADN : 40uL = 2,4 ug 4uL EcoRI, PstI, FastAp 12 uL FD buffer 56 uL water TOT =120uL
  • PCR2 ADN : 40uL = 1ug 6 uL EcoRI, PstI 12 uL FD buffer 64 uL water TOT= 128uL
  • 10 min at 37°C

    • Then PCR purification
  • Nanodrop : nanodrop : PCR 1 = 20,7 ng/uL ;PCR2 = 25,7

    • For the ligation :
  • Vector PCR1 100 ng = 5 uL; Insert PCR 2 300 ng = 12uL T4 ligase buffer 2uL ; T4 ligase 1 uL ; Total 20 uL
  • 20-40 min on the bench

  • Nanodrop : L= 270 ng/uL

    • Then I did a heat shock transformation (following the iGEM protocol : http://parts.igem.org/Help:Protocols/Transformation )
  • C + = 10 ng/uL of RFP in pS1C3, 2 uL transformed (10pg/ul RFP Control (pSB1C3 w/ BBa_J04450)
  • C - = nothing but efficients cells
  • T = Plasmid construct 2uL transformed
  • Each with 50 uL of chemically competents cells done yesterday.

    • Then I did 5 different plates :
  • 5 plates with 25ug/mL chloramphenicol in LB
  • 200 uL control + , 20 uL control +
  • 200 uL T , 20 uL T
  • 200 uL C -
  • Incubation overnight at 37°C


    • July 31th

    Goal 1

    Check the transformations I've done yesterday

    Procedure

    According to my plates my competent cells are working, red colonies on the C+. Transformation of my construction didn’t worked, I’ll start again all the process : PCR, purify, digest, ligate, integrate (heat shock), overnight culture.

    Goal 2

    PCR my backbone and my insert

    Procedure

  • PCR1' = Backbone + 068 and 069 primers, before purify
  • PCR2' = FINAL + 068 and 069 primers, before purify
  • After PCR and after purification : 1'p = 66,5 2'p =90,5 ng/uL
  • The gel migration shows a bad result for PCR 1’ (Linearized pSB1C3)

    •

    August 1st

    Goal

    PCR my backbone and my insert again : 1" and 2"

    Procedure

    PCR then purify

    August 5th

    Goal

    Ligation/transformations (insertion of FINAL block from IDT in the pSB1C3 backbone, FINAL is my Riboswitch associated with an EGFP)

    Procedure

  • Used 3 different insert PCR products
  • Ligation after digestion and purification of PCR2’ = 94,6ng/uL , PCR2’’= 57,5 ng/uL , PCR2’’’=83,4 ng/uL ; Backbone pSB1C3 = 37,9ng/uL
    • ' " "'
    Insert 4 7 5
    Backbone 5 5 5
    Buffer 2 2 2
    Ligase 1 1 1
    Water 8 5 7
    TOTAL 20uL 20uL 20uL
    I did then a transformation using the iGEM protocol (Heat Shock) with 50uL of chemically competent cells + 3 uL of Ligation Product for each (pSB1C3-TOTAL)
  • C + : pSB1C3-RFP
  • C - : just competent cells

  • Plated on Cm 25 ug/mL
  • 5 plates : ‘,’’,’’’,C+,C-

    • Results

  • C- : nothing
  • C+ : lot of red colonies
  • ‘ : lot
  • ‘’ : lot
  • ‘’’ : lot
  • I should have done a control transformation with just the backbone


    • August 6th

    Goal 1

    Cultivate my transformants

    Procedure

  • There is red and white transformant colonies, all resistant to Cm 25
  • I selected some white colonies overnight culture
  • On plate LB Cm —> it grew (some red, maybe contamination)
  • On broth LB Cm —> it grew (some red, maybe contamination)
  • Inoculated at 4:00 pm in LB Cm 25 bacteria from a culture ( red —> RFP ) with different AdoCbl concentrations in order to detect the minimal amount of B12 viable for e.coli : 5; 10; 20; 40; 80; 160 ng/mL

    • Goal 2

    PCR the pSB1C3 backbone and my FINAL riboswitch construction

    Procedure

  • 2 PCR each
  • I1 and I2 using primers 150 and 151
  • LP1 and LP2 using primers 152 and 153
  • Purification then nano drop : good for I2 : 112.4 ng/uL

    • Results

    It shows a good length only for I2 at 1300 bp.

    Goal 3

    Do the PCR again

    Procedure

    Template Primers MasterMix Water TOTAL Annealing T°C Elongation time
    I3 2ng = 2 uL 1 uL 25 uL 22 uL 50uL 56°C 2 min
    I4 2ng = 2 uL 2 uL 25 uL 21 uL 50uL
    LP3 25ng = 1uL 1 uL 25 uL 23 uL 50uL
    LP4 25ng = 1uL 2 uL 25 uL 22 uL 50uL

    Results

    After PCR purify, nanodrop (ng/uL)
    I3 I4 LP3 LP4
    30.7 177.6 86.7 93.9
    Gel migration
    Very good for LP4, good for LP3
    Nothing for I3 and I4


    August 7th

    Goal 1

    Digestion of LP4 and I2

    Procedure

    LP4 I2
    EcoRI 4uL 4uL
    PstI 4uL 4uL
    FastAP 4uL -
    FD Buffer 12uL 8uL
    DNA 2 ug (90 ng/uL) —> 20 uL 2ug (100 ng/uL) —> 20 uL
    Water 76 uL 44 uL
    TOTAL 120uL 80uL
    New nano drop : LP4 = 87,4 ; I2 = 90,4 ng/uL
    Waited 15 min at 37°C
    After digestion and PCR purification, nanodrop :
    38.2 ng/uL for I2 (d. and p.)
    31.2 ng/uL for LP4 (d. and p.)

    Goal 2

    Ligation of LP4dp and I2dp

    Procedure

    L*
    Vector LP4 dp 100 ng (31.2 ng/uL) —> 3uL
    Insert I2 dp 300 ng (38.2 ng/uL) —> 8uL
    T4 L,Buffer 2uL
    T4 ligase 1uL
    Water 6uL
    TOTAL 20 uL
    Waited 40 min on the bench (25°C)

    Goal 3

    Transformation of L*

    Procedure

    C- C+ T*
    ChemicallyCompetent Cells 50 uL 50 uL 50 uL
    L* - - 3 uL
    pSB1C3 with RFP 100ng/uL - 3 uL -
    iGEM Transfo protocol
    90 sec heat shock
    On saturday they were growing but no fluorescence

    August 10th

    Goal 1

    Colony PCR my T*

    Procedure

    Pick up 16 colonies on my two T* plates
    Put some bacteria in PCR tube + 2 uL of 148 and 149 (iGEM verification forward and backward) ;
    10uM + 50 uL of Master Mix Phusion (last in the tubes) + 45 uL of water (first in the tube)
    Waiting for a 1577 bp fragment on a gel Gel migration with a GR 1kb

    It is quiet bad

    August 11th

    Goal 1

    Do a new PCR of FINAL (G-block) and of pSB1C3 associated with RFP

    Procedure

    Template Primers Master Mix Phusion Water Time of elongation (72°C) T°C annealing Lenght Primers Nanodrop after purify (ng/uL)
    LP1* 5ng 2uL 25uL QSP for 50uL 2 min 56°C 2078 152 - 153 68.3
    LP2* 10ng 2uL 25uL QSP for 50uL 2 min 56°C 2078 152 - 153 133.5
    LP3* 2ng 2uL 25uL QSP for 50uL 2 min 56°C 2078 152 - 153 127.8
    F1* 2ng 2uL 25uL QSP for 50uL 1 min 58°C 1314 150 - 151 98
    F2* 3ng 2uL 25uL QSP for 50uL 1 min 58°C 1314 150 - 151 102
    F3* 1ng 2uL 25uL QSP for 50uL 1 min 58°C 1314 150 - 151 58

    Results


    Gel migration wit 1kb GR

    Goal 2

    Digestion of pSB1C3 + RFP

    Procedure

    Backbone+RFP
    Plasmid (100ng/uL) 10uL
    EcoRI 2uL
    PstI 2uL
    Buffer 6uL
    Water 38uL
    FastAP 2uL
    TOTAL 60uL

    Results

    Gel migration : order = 1kb GR - Digested product x3

    Then I cuted the 2100 fragment and did the gel extraction protocol

    Goal 3

    Digestion of pSB1C3 linearized (LP1,2,3 *) and F1,2,3 *

    Procedure

    F2d LP2d LP3d
    ADN 10 uL 10 uL 10 uL
    EcorRI 2 uL 2 uL 2 uL
    PstI 2 uL 2 uL 2 uL
    Buffer 4 uL 6 uL 6 uL
    Water 22 uL 38 uL 38 uL
    FastAP _ 2uL 2uL
    TOTAL 40 60 60
    Then 30min at 37°C

    Goal 4

    Ligations and transformations

    Procedure

    L1 L2 L3
    Vector 100ng LP2d (40.1ng/uL),2uL LP3d (21.1ng/uL),4 uL Backbone from digestion + electrophoresis extraction (9.1ng/uL), 10 uL
    Insert 300 ng (8.1ng/uL) (F2d) 30 uL 20 uL 10 uL
    T4 ligase buffer 4 uL 4 uL 2 uL
    T4 ligase 2 uL 2 uL 1 uL
    Water 0 10 uL 0
    TOTAL 40 uL 40 uL 20 uL
    Waited 20 min on the bench after mixing to do the ligation
    Used 3 uL for the transformations with 50 uL of competent cells
    (iGEM protocol) Waited 15 min instead of 30 min after put the ligation product (on ice)
    Heat shock 1 min 30
    2 hours recovery
    Plated C- (just chemically competents cells Heat Shocked) C (LP2 dp ! no ligation) L1, L2, L3

    Results

    Grew on L1, L2, L3 ; nothing on the two others
    Number of colonies :
    L1 ++ (50)
    L2 ++ (50)
    L3 +++ (100)


    August 12th

    Goal 1

    Ligation of LP2 alone and transfo for a negative control

    Procedure

    Vector : LP2 (40.1ng/uL) 2 uL
    No insert
    T4 ligase buffer : 4 uL
    T4 ligase : 2 uL
    Water : 32 uL
    TOTAL : 40 uL
    Transfo : use 3 uL of ligated product
    Result after culture on LB + Cm : nothing for the C and lot of colonies for L2

    Goal 2

    Colony PCR on yesterday transformations plates

    Procedure

    Using primers 148 - 149 (10uM), 2 uL each
    Dream Taq Master Mix : 10 uL
    Water : 6uL
    TOTAL : 20uL
    Colony n° 1 2 3 4 5 6 7 8 9 10 11 12 13
    Comes from L2-1 L2-2 L2-3 L2-4 L2-5 L1-1 L1-2 L1-3 L1-4 L3-1 L3-2 L3-3 L3-4

    Results

    Nothing : ?
    350 bp : recircularization (expected : 320 bp )
    Expected = 1700 ! —> nothing


    August 13th

    Goal 1

    Digestion

    Procedure

    I will digest F1* this time and try to insert it in LP2* digested too
    F1*d LP2*d
    ADN 2 ug (60,6 ng/uL) = 35 uL 2 ug (108,3 ng/uL) = 20 uL
    EcorI 4 uL 4 uL
    PstI 4 uL 4 uL
    FastAP - 4 uL
    Buffer 8 uL 12 uL
    Water 29 uL 76uL
    TOTAL 80 uL 120 uL
    Then incubated 1 h at 37°C

    Goal 2

    Ligation

    Procedure

    L4 LC (control, no insert)
    Insert 195 ng (35 ng/uL) = 6 uL -
    Vector 100 ng (35ng/uL) = 3uL 3 uL
    Ligase 1 uL 1 uL
    Ligase Buffer 2 uL 2 uL
    Water 8 uL 14 uL
    TOTAL 20 uL 20 uL
    Then waited 40 min on the bench
    And transformed 2uL of each tube in 50 uL of chemically competent cells.
    Then plated 3 petri dish (Cm 25ng/uL : 100 uL of L4; 150 uL of L4; 100 uL of LC
    37°C overnight.

    Result

    Number of colonies : 100 uL of L4 = 200 ; 150 uL of L4 = 300 ; 100 uL of LC = 3 The control is showing that my T4 colonies might have insertions

    Goal 3

    4 colony PCR on 24 colonies from L2 done yesterday with the control

    Procedure

    Made 4 tubes with 1 mL of water and introduced 6 colonies per tube in order to be able to do more screening. Let the tubes on the bench 1 hour (osmotic shock), then centrifugation 5 min 14 K rpm. Then took 8 uL of each suspension and put it in 4 PCR tubes.
    PCR1 PCR2 PCR3 C
    Colonies 1-6 ;8 uL 7-12 ; 8 uL 13-18 ; 8 uL 19-24 ; 8 uL
    Master Mix 10 uL 10 uL 10 uL 10 uL
    Primers 148 - 149 1 uL x 2 1 uL x 2 1 uL x 2 1 uL x 2
    Water 6 uL 6 uL 6 uL 6 uL
    TOTAL 20 uL 20 uL 20 uL 20 uL
    Result = nothing on the electrophoresis migration.
    It might be a problem in the protocol, maybe 1mL is to much and it’s over diluted so not enough template ?
    Maybe the cells didn’t exploded ?


    August 14th

    Goal

    Colony PCR (16 colonies from T4)

    Procedure

    Suspended T4 colony in 20 uL of water 6 uL
    Primers 148 - 149 1 uL x2
    Master Mix Phusion 8 uL
    TOTAL 16 uL
    x 16
    Anealing 51°C
    Elongation time : 1:30 min
    35 cycles

    Selected colony 6 for overnight culture in LB broth + Cm25
    Also colony 5 for a control
    The electrophoresis shows a bad level of insertion ( It is strange considering that my control - had just 3 colonies growing )
    Why 2 bands in the 6th colony ? Maybe multiple plasmid insertion some with insertion, some without.
    What to do?
    Work on the 6th colony
    Change my restriction enzymes
    Use DpnI


    August 17th

    Goal

    Miniprep and digest colonie 6 and 5 for control

    Procedure

    Using Promega SV Miniprep Kit
    Nanodrop after
    6 = 55 ng/uL
    5 = 51 ng/uL
    Digestion : Hind III enzyme (1 digestion site in my construction, inside my insert)
    5 6
    Hind III 4 uL 4 uL
    FastAP 4 uL 4 uL
    FD buffer 8 uL 8 uL
    DNA (50ng/uL) 20 uL 20 uL
    Water 44 uL 44 uL
    TOTAL 80 uL 80 uL
    Mix, Incubate 30 min 37°C PCR purification

    August 18th

    Goal1

    New digestion

    Procedure

    F2d' F3d' LP1d' LP3d'
    DNA 55ng/uL = 20uL 76 ng/uL = 15uL 76 ng/uL = 15uL 82ng/uL = 15uL
    FastAP 2uL 2uL
    EcoRI 2uL 2uL
    XbaI 2uL 2uL
    SpeI 2uL 2uL 2uL 2uL
    FD buffer 4uL 4uL 6uL 6uL
    Water 12uL 17uL 33uL 33uL
    TOTAL 40uL 40uL 60uL 60uL
    Nanodrop after purify 41 ng/uL 21 ng/uL 16 ng/uL 68 ng/uL
    20 min at 37°C

    Goal2

    Ligation

    Procedure

    La (F2d’-LP1d’) La c Lb (F3d’-LP3d’) Lb c
    F2dp’ 6uL
    LP1dp’ 7uL 7uL
    F3dp’ 15uL
    LP3dp’ 2uL 2uL
    Ligase 1uL 1uL 1uL 1uL
    L buffer 2uL 2uL 2uL 2uL
    Water 4uL 10uL 15uL
    TOTAL 20uL 20uL 20uL 20uL
    Transformed 5uL in 50 uL of competent cells - Heat shock Then plate it

    August 19th

    Results

    Yesterday plates : La (250uL) : 50
    Lb (250uL) : 0
    La c (50uL) : 1 colony
    Lb c (50uL) : 1 colony
    Nothing but competent cells : NOTHING

    Goal

    Colony PCR

    Procedure

    Cells in 10 uL of water 2 uL
    Primers 1uL x 2
    MasterMixDreamTaq 10 uL
    Water 6 uL
    Total 20 uL
    Expected 1577bp

    Result

    Then m iniprep of 22, 23 (+24?), 24
    Also plated LB + Cm
    And liquid culture

    August 21th

    Goal

    Digestion of my mini prep from yesterday

    Procedure

    Nanodrop :
    22 = 23.5ng/uL
    23 = 53.7ng/uL
    24 = 25.5ng/uL
    22 23 24
    DNA 16 16 16
    Buffer 2 2 2
    Hind III 1 1 1
    Nco I 1 1 1
    TOTAL 20 20 20
    Expected length 2040 bp (no insertion, recircularisation) 1315 and 2026 bp (insertion) 2040 bp
    37°C 20 min
    Gel order : GR1kb , 22, 23, 24, GR1kb

    Exactly what I was expecting
    Band 23 : 3700 = plasmid undigested ??

    New gel
    Order : GR 1kb, 23, 23 d, 24, 24d

    PS : 23 is two bands now maybe because the digestion is now finished (it was on the bench for one hour)
    23 sent to sequencing

    August 31th

    Goal 1

    PCR for my Gibson 1 = Replace the RBS and first codon of the RFP in the classical pSB1C3 associated with Part:BBa J04450 by the riboswitch sequence

    Procedure

    PCR
    Insert Backbone
    Template 1ng = 2uL 1 uL 1ng = 2uL 1uL
    Primers 209-210
    1uL    		 210-211
    1uL
    MasterMix 25 uL 25 uL
    Water 23 uL 24 uL 23 uL 24 uL
    TOTAL 50uL 50uL
    Elongation time 30 sec 2 min
    Expected length 243 3149

    Results

    Gel order : GR 1kb, B1, B2, I1, I2

    Bad result for B1 , B2

    Goal 2

    New PCR for the backbone

    Procedure

    B2 B3 B4
    Template 3uL (ng) 3uL (ng) 3uL (ng)
    MasterMixPhusion 25 uL 25 uL 25 uL
    DMSO 1uL 1uL 1uL
    Primers 1uL 1uL 1uL
    Water 20 uL 20 uL 20 uL
    T°C annealing 56°C 58°C 60°C

    Result


    So bad

    September 1st

    Goal 1

    PCR FINAL for a new transformation

    Procedure

    F1 F2 F3
    Template 2ng 5ng 10ng
    Primers 150-151 2uL 2uL 2uL
    MasterMix 25uL 25 uL 25 uL
    Water 19 uL 16uL 11uL
    TOTAL 50 uL 50 uL 50 uL
    T°C gradient : 0 - 59°C
    1 - 58.8°C
    2 - 58.4°C
    3 - 57.9°C
    4 - 57.2°C
    5- 56.6°C

    Result

    Gel order : GR1KB-10-11-12-13-14-15- GR1KB-20-21-22-23-24-25- GR1KB-30-31-32-33-34-35

    Goal 2

    Gibson 1 - New PCR

    Procedure

    B5 B6 B7 I3 I4 I5
    Template 1ng 3ng 6ng 1ng 3ng 6ng
    Primers 2uL 2uL 2uL 2uL 2uL 2uL
    MasterMixPhusion 25uL 25uL 25uL 25uL 25uL 25uL
    Water 20 uL 20 uL 20 uL 20 uL 20 uL 20 uL
    TOT 50 uL 50 uL 50 uL 50 uL 50 uL 50 uL
    Primers used 211-212 209-210
    T°C annealing 69°C 66°C
    Elongation time 2min 15sec

    Results

    Gel order : - - - B5 B6 B7 I4 I5 ( I3 was lost )

    September 2nd

    Goal 1

    Digestion/Ligation

    Procedure

    The PCR product was purified before.
    Digestion :
    F11d pSB1C3d
    DNA 1:3 55ng/uL, 1.5 ug —> 30 uL 96 ng/uL, 0.75 ug —> 8 uL
    Fast AP 2uL
    EcoRI 2uL 2uL
    SpeI 2uL 2uL
    FD Buffer 4uL 6uL
    Water 2uL 40uL
    TOTAL 40uL 60uL
    Nanodrop after purif 8.6 11 (after gel extract)


    I extracted the 3kb band from the gel
    Ligation :
    La (F2d’-LP1d’) La c
    F11d 25 uL
    pSB1C3d 10uL 10uL
    Ligase 1uL 1uL
    L buffer 4uL 4uL
    Water 25uL
    TOTAL 40uL 40uL
    Then on the bench for 1 hour

    Goal 2

    Back to my Gibson 1

    Procedure

    ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are usually fine.
    G11 G12
    B5 100ng — > 3uL 100ng — > 3uL
    I5 7.7ng (equimolar) —> 1uL
    I2 7.7ng (equimolar according to NEB Bio Calculator) —> 1uL
    GibsonMix 15uL 15uL
    Water 1 uL 1 uL
    TOTAL 20uL 20uL
    Then 60 min to 50°C
    Then I did a transformation by Heat Shock using 2 uL of the product and I plated it.

    September 3rd

    Result

    Result of my plating from yesterday
    G11: 200uL —> 300 colonies (3 were red)
    20 uL —> 40 colonies (2 red)
    G12 : 200uL —> 30 colonies (9 were red)
    20uL —> 3 colonies (1 red)

    Goal 1

    Colony PCR (on colonies that don’t show any red fluo)

    Procedure

    16 colonies : Master Mix dream Taq 10uL , Primers Fwd 148 1uL , Primer Rv 149 1uL, Water 8uL , TOTAL 20uL
    1 min 45 elongation expected length 1566; T°Cannealing 52°C; 95°C 8min first

    Result

    Colony 1 to 16, GR 1kb between 8 and 9

    Liquid culture in M9 (without B12) with Cm 25 for colonies 3, 4, 5, 7, 8, 9 , 10

    Goal 2

    Colony PCR on the red colonies

    Procedure

    Master Mix dream Taq 10uL , Primers Fwd 148 1uL , Primer Rv 149 1uL, Water 8uL , TOTAL 20uL

    Result



    Colony 11’ is quiet interesting, the 3' too
    Liquid culture in M9 Cm25 of 1’, 2’, 3’, 11’ without B12 and with 5 mg/L of adenosylcobalamin and in LB Cm25

    September 4th

    Goal 1

    PCR my new G-block for Gibson 2

    Procedure

    R (Riboswitch) B (pSB1C3)
    Template DNA 2ng 2ng
    Primers 1 (10uM) 2uL (213) 2uL (215)
    Primers 2 (10uM) 2uL (214) 2uL (216)
    Phusion Master Mix 25 uL 25 uL
    Water 19 uL 19 uL
    TOTAL 50 uL 50 uL
    T°C annealing 67°C 66°C
    Time elongation 25 sec 1 min 10
    Length 476 3149

    Result



    Nothing